Glycoconjugates and their use as potential vaccines against infection by Shigella flexneri

ABSTRACT

A conjugate molecule comprising an oligo- or polysaccharide covalently bound to a carrier and its use as potential vaccine against infection by  S. Flexneri.

FIELD OF THE INVENTION

This invention relates to compositions and methods for eliciting animmunogenic response in mammals, including responses that provideprotection against, or reduce the severity of bacterial infections. Moreparticularly it relates to the use of oligo- or polysaccharides obtainedfrom natural sources and/or through synthesis or recombinant technology,and conjugates thereof to induce serum antibodies having protectiveactivity against Shigella flexneri, in particular S. flexneri serotype2a. These saccharides and/or conjugates thereof are useful as vaccinesto induce serum antibodies which have protective activity against S.flexneri, in particular S. flexneri type 2a, and are useful to preventand/or treat shigellosis caused by S. flexneri.

The present invention also relates to diagnostic tests for shigellosisusing one or more of the oligo- or polysaccharides, conjugates orantibodies described above.

BACKGROUND OF THE INVENTION

Since the discovery of Shigella dysenteriae type 1 (Shiga's bacillus)more than a century ago (R. Shields and W. Burnett, Zentl. Bakterio.,1898, 24, 817-828), shigellosis or bacillary dysentery has been known asa serious infectious disease, occurring in humans only (T. G. Keusch andM. L. Bennish, Shigellosis, Plenum Medical Book Company, New York, 1991,p. 593-620). In a recent survey of the literature published between 1966and 1997 (K. L. Kotloff, J. P. Winickoff, B. Ivanoff, J. D. Clemens, D.L. Swerdlow, P. J. Sansonetti, G. K. Adak and M. M. Levine, Bull. WHO,1999, 77, 651-666), the number of episodes of shigellosis occurringannually throughout the world was estimated to be 164.7 million, ofwhich 163.2 million were in developing countries. Up to 1.1 millionannual deaths were associated with shigellosis during the same period.Occurrence of the disease is seen as a correlate of sanitary conditions,and those are not likely to improve rapidly in areas at risk.

The financial status of the populations in which shigellosis exists inits endemic forms, as well as the emerging resistance to antimicrobialdrugs (M. U. Khan, Int. J. Epidemiol., 1985, 14, 607-613; B. A.Iwalokun, G. O. Gbenle, S. I. Smith, A. Ogunledun, K. A. Akinsinde andE. A. Omonigbehin, J. Health Popul. Nutr., 2001, 19, 183-190), limit theimpact of the latter. Of the four species of Shigellae, S. flexneri isthe major responsible for the endemic form of the disease, with serotype2a being the most prevalent. The critical importance of the developmentof a vaccine against Shigellae infections was first outlined in 1987(World Health and Organization, Bull. W.H.O., 1987, 65, 17-25). Due toincreasing resistance of all groups of Shigellae to antibiotics (S.Ashkenazi, M. May-Zahav, J. Sulkes and Z. Samra, Antimicrob. AgentsChemother., 1995, 39, 819-823) vaccination remained a high priority asstated by the World Health Organization ten years later (WHO, WeeklyEpidemiol. Rec., 1997, 72, 73-79). In the meantime, several experimentalvaccines have gone through field evaluation (T. S. Coster, C. W. Hoge,L. L. van der Verg, A. B. Hartman, E. V. Oaks, M. M. Venkatesan, D.Cohen, G. Robin, A. Fontaine-Thompson, P. J. Sansonetti and T. L. Hale,Infect. Immun., 1999, 67, 3437-3443; J. H. Passwell, E. Harlev, S.Ashkenazi, C. Chu, D. Miron, R. Ramon, N. Farzan, J. Shiloach, D. A.Bryla, F. Majadly, R. Roberson, J. B. Robbins and R. Schneerson, Infect.Immun., 2001, 69, 1351-1357) but there are as yet no licensed vaccinesfor shigellosis.

Shigella's lipopolysaccharide (LPS) is a major surface antigen of thebacterium. The corresponding O—SP domain (O—SP) is both an essentialvirulence factor and the target of the infected host's protective immuneresponse (D. Cohen, M. S. Green, C. Block, T. Rouach and I. Ofek, J.Infect. Dis., 1988, 157, 1068-1071; D. Cohen, M. S. Green, C. Block, R.Slepon and I. Ofek, J. Clin. Microbiol., 1991, 29, 386-389). Indeed,using the pulmonary murine model for shigellosis, it was demonstratedthat the presence locally, preliminary to infection, of a secretoryantibody of isotype A specific for an epitope located on the O—SP moietyof the LPS of S. flexneri 5a, prevented any host homologous infection(A. Phalipon, M. Kauffmann, P. Michetti, J.-M. Cavaillon, M. Huerre, P.Sansonetti and J.-P. Krahenbuhl, J. Exp. Med., 1995, 182, 769-778).Based on the former hypothesis that serum IgG anti-LPS antibodies mayconfer specific protection against shigellosis (J. B. Robbins, C. Chuand R. Schneerson, Clin. Infect. Dis., 1992, 15, 346-361), severalpolysaccharide-protein conjugates, targeting either Shigella sonnei, S.dysenteriae 1 or S. flexneri serotype 2a, were evaluated in humans (J.H. Passwelle, E. Harlev, S. Ashkenazi, C. Chu, D. Miron, R. Ramon, N.Farzan, J. Shiloach, D. A. Bryla, F. Majadly, R. Roberson, J. B. Robbinsand R. Schneerson, Infect. Immun., 2001, 69, 1351-1357; D. N. Taylor, A.C. Trofa, J. Sadoff, C. Chu, D. Bryla, J. Shiloach, D. Cohen, S.Ashkenazi, Y. Lerman, W. Egan, R. Schneerson and J. B. Robbins, Infect.Immun., 1993, 61, 3678-3687). In the case of S. sonnei, recent fieldtrials allowed Robbins and co-workers to demonstrate the efficacy of avaccine made of the corresponding detoxified LPS covalently linked torecombinant exoprotein A (D. Cohen, S. Ashkenazi, M. S. Green, M.Gdalevich, G. Robin, R. Slepon, M. Yavzori, N. Orr, C. Block, I.Ashkenazi, J. Shemer, D. N. Taylor, T. L. Hale, J. C. Sadoff, D.Pavliovka, R. Schneerson and J. B. Robbins, The Lancet, 1997, 349,155-159). Conversion of polysaccharide T-independent antigens toT-dependent ones through their covalent attachment to a carrier proteinhas had a tremendous impact in the field of bacterial vaccines. Severalsuch neoglycoconjugate vaccines are currently in use against Haemophilusinfluenzae b (R. W. Ellis and D. M. Granoff, Development and clinicaluse of Haemophilus b conjugate vaccines, Dekker, New York, 1994),Neisseria meningitidis (P. Richmond, R. Borrow, E. Miller, S. Clark, F.Sadler, A. Fox, N. Begg, R. Morris and K. Cartwright, J. Infect. Dis.,1999, 179, 1569-1572) and Streptococcus pneumoniae (M. B. Renels, K. M.Edwards, H. L. Keyserling, K. S. Reisinger, D. A. Hogerman, D. V.Madore, I. Chang, P. R. Paradiso, F. J. Malinoski and A. Kimura,Pediatrics, 1998, 101, 604-611). These polysaccharide-protein conjugatevaccines are highly complex structures, whose immunogenicity depends onseveral parameters amongst which are the length and nature of thesaccharide component as well as its loading on the protein. It isreasonably admitted that control of these parameters is somewhatdifficult when dealing with polysaccharides purified from bacterial cellcultures. As recent progress in carbohydrate synthesis allows access tocomplex saccharides, it has been suggested that the use of well-definedsynthetic oligosaccharides may allow a better control, and consequentlythe optimisation, of these parameters. Indeed, available data on S.dysenteriae type 1 indicate that neoglycoconjugates incorporating di-,tri- or tetramers of the O—SP repeating unit were more immunogenic thana detoxified LPS-human serum albumin conjugate of reference (V. Pozsgay,C. Chu, L. Panell, J. Wolfe, J. B. Robbins and R. Schneerson, Proc.Nail. Acad. Sci. USA, 1999, 96, 5164-5197).

Besides, recent reports demonstrate that short oligosaccharidescomprising one repeating unit may be immunogenic in animal models (B.Benaissa-Trouw D. J. Lefeber, J. P. Kamerling, J. F. G. Vliegenthart, K.Kraaijeveld and H. Snippe, Infect. Immun., 2001, 69, 4698-4701; F.Mawas, J. Niggemann, C. Jones, M. J. Corbet, J. P. Kamerling and J. F.G. Vliegenthart, Infect. Immun., 2002, 70, 5107-5114). Another criticalparameter in the design of neoglycoconjugate vaccines is the carrierprotein. As potential applications for these vaccines are expanding, theneed for new carrier proteins licensed for human use is growing (J. B.Robbins, R. Schneerson, S. C. Szu and V. Pozsgay inPolysaccharide-protein conjugate vaccines, vol. (S. Plotkin and B.Fantini Eds), Elsevier, Paris, 1996, pp. 135-143). That syntheticpeptides representing immunodominant T-cell epitopes could act ascarriers in polysaccharide and oligosaccharide conjugates has beensuggested (G. J. P. H. Boons, P. Hoogerhout, J. T. Poolman, G. A. vander. Marel and J. H. van Boom, Bioorg. Med. Chem., 1991, 1, 303-308) andlater on demonstrated (E. Lett, S. Gangloff, M. Zimmermann, D. Wachsmannand J.-P. Klein, Infect. Immun., 1994, 62, 785-792; A. Kandil, N. Chan,M. Klein and P. Chong, Glycoconjugate J., 1997, 14, 13-17). Besides, theuse of T-cell epitopes offers several advantages, including potentialaccess to well-defined conjugates with no risk of epitopic suppression,as this latter phenomenon appeared to be a major drawback of proteincarriers (T. Barington, M. Skettrup, L. Juul and C. Heilmann, Infect.Immunol., 1993, 61, 432-438; M.-P. Schutze, C. Leclerc, M. Jolivet, F.Audibert and L. Chedid, J. Immunol., 1985, 135, 2319-2322). Polypeptidescontaining multiple T-cell epitopes have been generated in order toaddress the extensive polymorphism of HLA molecules (P. R. Paradiso, K.Dermody and S. Pillai, Vaccine Res., 1993, 2, 239-248). In otherstrategies, universal T-helper epitopes compatible with human use havebeen characterized, for example from tetanus toxoid (D. Valmori, A.Pessi, E. Bianchi and G. P. Corradin, J. Immunol., 1992, 149, 717-721),or engineered such as the pan HLA DR-binding epitope (PADRE) (J.Alexander, J. Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal,K. Snoke, H. M. Serra, R. T. Kubo, A. Sette and H. M. Grey, Immunity,1994, 1, 751-761). Recently, covalent attachment of the human milkoligosaccharide, lacto-N-fucopentose II, to PADRE resulted in a linearglycopeptide of comparable immunogenicity to that of a glycoconjugateemploying human serum albumine (HAS) as the carrier (J. Alexander, A.-F.d. Guercio, A. Maewal, L. Qiao, J. Fikes, R. W. Chesnut, J. Paulson, D.R. Bundle, S. DeFrees and A. Sette, J. Immunol., 2000, 164, 1625-1633).

Based on these converging data, the inventors have focused on thedevelopment of well-defined neoglycoconjugate as an alternative topolysaccharide protein conjugate vaccines targeting infections caused byS. flexneri serotype 2a. The target neoglycoconjugates were constructedby covalently linking an immunocarrier, serving as T-helper epitope(s),to appropriate carbohydrate (oligo- or polysaccharide) haptens, servingas B epitopes mimicking the S. flexneri 2a O—Ag. To this end, arationale approach involving a preliminary study of the interactionbetween the bacterial O—SP and homologous protective monoclonalantibodies, was employed to define the carbohydrate haptens.

SUMMARY OF THE INVENTION

Abbreviation: LPS: lipopolysaccharide; O—SP: O-specific polysaccharide;TT: tetanus toxoid; DCC: dicyclohexyl carbodiimide; Rhap:rhamnopyranosyl; Glcp: glucopyranosyl; GlcNAcp:2-acetamido-2-deoxy-glucopyranosyl.

In the instant invention, the list of polysaccharides designated L1consists of:

(X)_(x)-{B(E)C}—(Y)_(y)

(X)_(x)-{(E)CD}-(Y)_(y)

(X)_(x)-{AB(E)C}—(Y)_(y)

(X)_(x)-{B(E)CD}-(Y)_(y)

(X)_(x)-{(E)CD)A}-(Y)_(y)

(X)_(x)-{DAB(E)C}n-(Y)_(y)

(X)_(x)-{B(E)CDA}n-(Y)_(y)

(X)_(x)-{(E)CDAB}n-(Y)_(y)

(X)_(x)-{AB(E)CD}n-(Y)_(y)

(X)_(x)-{DAB(E)CD}-(Y)_(y)

(X)_(x)-{B(E)CDAB(E)C}—(Y)_(y)

wherein:

A is an alphaLRhap-(1,2) residue

B is an alphaLRhap-(1,3) residue

C is an alphaLRhap-(1,3) residue

E is an alphaDGlcp-(1,4) residue

D is a betaDGlcNAcp-(1,2) residue

x and y are independently selected among 0 and 1

X and Y are independently selected among A, B, C, D, E, AB, B(E), (E)C,CD, DA, AB(E), B(E)C, (E)CD, CDA, AB(E)C, B(E)CD, (E)CDA, CDAB, DAB(E)and wherein n is an integer comprised between 1 and 10 covalently boundto a carrier.

Saccharides selected from the group consisting of:

{B(E)CD}

{(E)CDAB}n

{AB(E)CD}n

wherein A, B, C, D, E and n have the same meaning as above are new andare another object of the invention.

It is an object of the present invention to produce an antigen based onnatural, modified-natural, synthetic, semi-synthetic or recombinantoligo- or polysaccharides which have subunits, selected from the listL1. Preferably, these oligo- or polysaccharides of the invention areantigenically similar to an antigenic determinant of the O—SP of S.flexneri type 2a which contains [AB(E)CD] subunits. It is also an objectof the invention to provide molecules, for example oligo- orpolysaccharides, which are structurally related and/or antigenicallysimilar to those oligo- and polysaccharides from the list L1. The oligo-or polysaccharides may be conjugated to an immunocarrier to formconjugates. These conjugates thereof are immunogenic and elicit serumantibodies that are protective against S. flexneri, in particular S.flexneri type 2a and which are useful in the prevention and treatment ofshigellosis caused by S. flexneri. These oligo- or polysaccharides andconjugates thereof, and the antibodies which they elicit, are alsouseful for studying S. flexneri, in particular S. flexneri type 2a, invitro or its products in patients. The oligo- or polysaccharides mayalso be conjugated to other carriers which are suitable for labelling orimmobilizing said oligo- or polysaccharides on a solid phase.

It is yet another object of the present invention to provide animmunogen that elicits antibodies which are protective against S.flexneri, in particular S. flexneri type 2a and which react with, orbind to the O—SP of S. flexneri type 2a, wherein the immunogen is basedon a natural, modified natural, synthetic, semi-synthetic or recombinantoligo- or polysaccharide containing one or more subunits selected fromthe list L1 or a structurally related, immunologically similar, oligo-or polysaccharide, and/or conjugate thereof.

It is yet another object of the present invention to provide antibodieswhich have protective activity against S. flexneri, in particular S.flexneri type 2a, and which react with, or bind to the O—SP of S.flexneri type 2a, wherein the antibodies may be elicited by immunizationwith a natural, modified natural, or synthetic oligo- or polysaccharidecontaining subunits from the list L1 or a structurally relatedimmunologically similar, oligo- or polysaccharide, and/or conjugatethereof.

It is yet another object of the present invention to provide oligo- orpolysaccharides or conjugates thereof with a carrier which are useful asvaccines to prevent and/or treat shigellosis.

It is yet another object of the present invention to prepare antibodiesfor the treatment of established shigellosis. Antibodies elicited by themolecules of the invention are able to provide passive protection to anindividual exposed to S. flexneri, in particular S. flexneri type 2a, toprevent, treat, or ameliorate infection and disease caused by themicroorganism.

It is yet another object of the present invention to provide diagnostictests and/or kits for shigellosis caused by S. flexneri, in particularS. flexneri type 2a, using one or more of the oligo- or polysaccharides,conjugates, or antibodies of the present invention.

It is yet another object of the present invention to provide an improvedmethod for synthesizing an oligo- or polysaccharide containing one ormore subunits or the list L1.

According to the present invention, methods are provided to isolate,substantially purify and/or synthesize natural, modified-natural,synthetic, semi-synthetic or recombinant oligo- or polysaccharidescontaining subunits of the L1 list or structurally related,immunologically similar, oligo- or polysaccharides. Preferably, theseoligo- and polysaccharides are structurally related and/orimmunologically similar to an antigenic determinant of the O—SP of S.flexneri type 2a.

Methods are also provided to conjugate the natural, modified-natural,synthetic, semi-synthetic or recombinant oligo- or polysaccharide of theinvention with a carrier.

DETAILED DESCRIPTION OF THE INVENTION

Oligo- and Polysaccharides:

This invention provides a synthetic, semi-synthetic, natural,modified-natural or recombinant oligo- or polysaccharide containingsubunits from the list L1.

Methods for synthesizing S. flexneri 2a di- tri-, tetra, penta andoctasaccharides are know from the prior art (F. Segat Dioury et al.,Tetrahedron Asymmetry, 13, 2002, 2211-2222; C. Costachel et al., J.Carbohydrate Chemistry, 19(9) 2000, 1131-1150; L. Mulard et al., J.Carbohydrate Chemistry, 19(7), 2000, 849-877 F. Belot et al.,Tetrahedron Letters, 43, 2002, 8215-8218; L. Mulard et al., Tetrahedron58, 2002, 2593-2604; L. Mulard et al., J. Carbohydrate Chemistry, 19(2),2000, 193-200).

An improved method to synthesize oligo- or polysaccharides is set forthin the examples below. Notably the synthesis of a decasaccharide wasperformed by condensation of two pentasaccharide intermediates.

DEFINITIONS

“oligosaccharide” as defined herein, is a carbohydrate containing fromtwo to twenty monosaccharide units linked together, “oligosaccharide” isused herein in a liberal manner to denote the saccharides describedherein; this usage differs from the standard definition thatoligosaccharides may contain up to and including ten monosaccharideunits (Joint Commission on Biological Nomenclature, Eur. J. Biochem.1982, 126, 433-437).

“polysaccharide” as defined herein, is a carbohydrate containing morethan twenty monosaccharide subunits linked together.

“structurally-related” oligo- or polysaccharide” as defined herein, is amodified oligo- or polysaccharide from the list L1, characterized by itsability to immunologically mimic the antigenic determinant of the O—SPof S. flexneri, in particular S. flexneri type 2a. Such modified oligo-or polysaccharide can be obtained by structure alterations that renderthe modified polysaccharide antigenically similar to the antigenicdeterminant of the O—SP of S. flexneri 2a. Such a modified oligo- orpolysaccharide can be obtained, for example, by means of a specificspacer constraining said oligosaccharide into the conformation it bearsin the native O—SP.

“immunoreact” means specific binding between an antigenicdeterminant-containing molecule and a molecule containing an antibodycombining site such as a whole antibody molecule or a portion thereof.

“antibody” refers to immunoglobulin molecules and immunologically activeor functional fragments of immunoglobulin molecules comprising anantigen recognition and binding site. Exemplary antibody molecules areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules and active fragments of an immunoglobulin molecule, includingthose portions known in the art as Fab, Fab′, F(ab′)₂ and scFv, as wellas chimeric antibody molecules.

“immunologically similar to” or “immunologically mimic” refers to theability of an oligo- or polysaccharide of the invention to immunoreactwith, or bind to, an antibody of the present invention that recognizesand binds to a native antigenic determinant on the O—SP of S. flexneritype 2a.

functional group refers to groups of atoms characterized by theirspecific elemental composition and connectivity. Said functional groupsconfer reactivity upon the molecule that contains them. Commonfunctional groups include: Primary amines: R—NH₂; Primary Imines:—C(═NH)—R′; Azo: [Azo, —N═N—R′; Nitrile, —C≡N; Carboxylic acid,Carboxyl: —C(═O)OH), carboxylic acid and derivatives thereof like ester:—C(═O)O—R′ or activated ester; Carbonyl: [Aldehyde: —C(═O)H; Ketone,—C(═O)—R′], or derivatives thereof as masked carbonyl such as acetal orthioacetal; Alkenes: —CH═CH—R′; Alkynes: —C≡C—R′; Isocyanates: —N═C═O;Isothiocyanate: —N═C═S; Thioacyl —SCO—R′, Thiol —SH, dithiol: —S—S—R′;Azide —N₃: Hydrazide: —CONHNH₂, Hydrazine, Maleimide, O-alkylhydroxylamine, halogen,

“carrier” refers to any molecule which can be covalently bound to anoligo- or polysaccharide of the invention to form the glycoconjugate ofthe invention. It includes immunocarriers for use as vaccine and othercarriers for preparing diagnostic reagents.

“immunocarrier” refers to an immunogenic molecule or a fragment of amolecule which is recognized by T cells and is able to induce anantibody response.

“other carriers for preparing diagnostic reagents” refers to agentscommonly used to immobilize molecules onto a solid phase or to labelmolecules.

“a label” refers to any substance which can produce a signal which canbe detected by any appropriate mean.

“glycoconjugate” refers to an oligo- or polysaccharide from the list L1covalently bound to a carrier.

“prevention and treatment” refers to the prevention of infection orreinfection, reduction or elimination of the symptoms, and reduction orcomplete elimination of the pathogen. Treatment may be effectedprophylactically (prior to infection) or therapeutically (followinginfection).

Oligo- and Polysaccharide Conjugates (Glycoconjugate)

The oligo- or polysaccharides of the invention can be bound covalentlyto a protein or peptide carrier. This covalent bond can be a direct bondbetween the oligo- or polysaccharide and the peptide or protein.

According to another variant, the oligo- or polysaccharide of the L1list can be linked to the protein or peptide via a spacer molecule. Theoligo or polysaccharide can be functionalized by an —O—R—Z group,wherein R is an alkyl group comprising 1 to 12 carbon atoms, preferably1 to 6 carbon atoms, preferably an ethyl group, and Z is a functionalgroup which reacts with a functional group of the protein or peptidecarrier. Preferably Z is —NH₂.

The oligo and polysaccharide of the list L1 bearing an —O-alkyl-Z andpreferably those bearing an —O-alkyl-NH₂ spacer molecule are anotherobject of the instant invention.

Notably molecules:

{B(E)C}—O—R—NH₂

{(E)CD}-O—R—NH₂

{AB(E)C}—O—R—NH₂

{B(E)CD}-O—R—NH₂

{(E)CDA}-O—R—NH₂

{DAB(E)C}n-O—R—NH₂

{B(E)CDA}n-O—R—NH₂

{(E)CDAB}n-O—R—NH₂

{AB(E)CD}n-O—R—NH₂

{DAB(E)CD}-O—R—NH₂

{B(E)CDAB(E)C}—O—R—NH₂

wherein A, B, C, D, E and n have the same meaning as above are ofspecial interest.

The oligo- or polysaccharide functionalized by an —O-alkyl-NH₂ group, isthen transformed in manner known to the man skilled in the art in an—O-alkyl-NH—CO—CH₂—R′, wherein —R′ is selected among a S-acetyl group, alinear haloalkyl group having from 1 to 7, and preferably 1 to 3 atomsof carbone, and preferably wherein the halogen is Br, and linearcarboxylic acid group having preferably 2 to 3 atoms of carbon, Forexample, the functionalized oligo- or polysaccharides with a S-acetylgroup can be deprotected resulting in the free thiol to be reacted witha carrier which is functionalized by a haloacetyl or a maleimide group.Another strategy consists in establishing a bond between the oligo- orpolysaccharide and the protein or peptide via a spacer bearing aβ-alanine.

According to another variant of the invention, oligo- andpolysaccharides of the L1 list are terminated by an —OQ group, wherein Qis selected among alkyl and alkenyl groups comprising 1 to 12 carbonatoms. Preferably Q is selected among methy and allyl. Particularly, asaccharide derivative selected from the group consisting of:

{B(E)CD}-OQ

{(E)CDAB}n-OQ

{AB(E)CD}n-OQ

{DAB(E)C}m-OQ

{B(E)CDA}m-OQ

{DAB(E)CD}-OQ

wherein A, B, C, D, E and n have the same meaning as above and m iscomprised from 2 and 10.

Methods for binding oligo- and/or polysaccharides to a non-toxicnon-host protein are well known in the art. For example, in U.S. Pat.No. 5,204,098 and U.S. Pat. No. 5,738,855 it is taught that an oligo- orpolysaccharide containing at least one carboxyl group, throughcarbodiimide condensation, may be thiolated with cystamine, or aminatedwith adipic dihydrazide, diaminoesters, ethylenediamine and the like.Groups which could be introduced by the method, or by other methodsknown in the art, include thiols, hydrazides, amines and carboxylicacids. Both the thiolated and the aminated intermediates are stable, maybe freeze dried, and stored in cold. The thiolated intermediate may bereduced and covalently linked to a polymeric carrier containing adisulfide group, such as a 2-pyridyldithio group. The aminatedintermediate may be covalently linked to a polymeric carrier containinga carboxyl group through carbodiimide condensation.

The oligo- or polysaccharide can be covalently bound to a carrier withor without a linking molecule. To conjugate without a linker, forexample, a carboxyl-group-containing oligo- or polysaccharide and anamino-group-containing carrier are mixed in the presence of a carboxylactivating agent, such as a carbodiimide, in a choice of solventappropriate for both the oligo- or polysaccharide and the carrier, as isknown in the art (Szu, S. C., A. L. Stone, J. D. Robbins, R. Schneerson,and J. B. Robbins, 1987, Vi capsular polysaccharide-protein conjugatesfor prevention of typhoid fever. J. Exp. Med., 166:1510-1524). Theoligo- or polysaccharide is preferably conjugated to a carrier using alinking molecule. A linker or crosslinking agent, as used in the presentinvention, is preferably a small linear molecule having a molecularweight of approximately <500 daltons and is non-pyrogenic and non-toxicin the final product form.

To conjugate with a linker or crosslinking agent, either or both of theoligo- or polysaccharide and the carrier may be covalently bound to alinker first. The linkers or crosslinking agents are homobifunctional orheterobifunctional molecules, (see references provided in BioconjugateTechniques, G. T. Hermanson, Ed, Academic Press San Diego, 1995). e.g.,adipic dihydrazide, ethylenediamine, cystamine, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-[N-(2-iodoacetyl)-β-alanyl]propionate-propionate (SIAP),succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC),3,3′-dithiodipropionic acid, and the like. Among the class ofheterobifunctional linkers are omega-hydroxy alkanoic acids.

According to the type of bonding between the oligo- or polysaccharideand the carrier, there is the possibility of preparing a conjugatemolecule wherein the ratio of the oligo- or polysaccharide versus thecarrier can vary between 1:1 and 30:1. Preferably, this ratio iscomprised between 5:1 and 20:1.

A carrier can be a natural, modified-natural, synthetic, semi-syntheticor recombinant material containing one or more functional groups, forexample primary and/or secondary amino groups, azido groups, or carboxylgroup. The carrier can be water soluble or insoluble. Carriers thatfulfil these criteria are well-known to those of ordinary skill in theart.

Immunocarriers are chosen to increase the immunogenicity of the oligo-or polysaccharide and/or to raise antibodies against the carrier whichare medically beneficial.

Suitable immunocarriers according to the present invention includeproteins, peptides, polysaccharides, polylactic acids, polyglycolicacids, lipid aggregates (such as oil droplets or liposomes), andinactivated virus particles.

According to an advantageous embodiment of the glycoconjugate moleculeof the invention, it is covalently bound to a protein or a peptidecomprising at least one T-helper cell epitope, for use as a vaccineagainst S. flexneri infection.

Protein carriers known to have potent T-cell epitopes, include but arenot limited to bacterial toxoids such as tetanus, diphtheria and choleratoxoids, Staphylococcus exotoxin or toxoid, Pseudomonos aeruginosaExotoxin A and recombinantly produced, genetically detoxified variantsthereof, outer membrane proteins (OMPs) of Neisseria meningitidis andShigella flexneri proteins. The recombinantly-produced, non-toxic mutantstrains of Pseudomonos aeruginosa Exotoxin A (rEPA) are described inFattom et al., Inf. Immun., 1993, 61, 1023-1032. The CMR 197 carrier isa well characterized non-toxic diphtheria toxin mutant that is useful inglycoconjugate vaccine preparations intended for human use (Bixler etal., Adv. Exp. Med. Biol., 1989, 251, 175-; Constantino et al. Vaccine,1992). Other exemplary protein carriers include the Fragment C oftetanus toxin, and the Class 1 or Class 2/3 OMPs. Also CRM 9 carrier hasbeen disclosed for human immunisation. (Passwell J H et al. PediatrInfect Dis J. (2003) 22, 701-6).

Synthetic peptides representing immunodominant T-cell epitopes car alsoact as carriers in polysaccharide and oligosaccharide conjugates. Thepeptide carriers include polypeptides containing multiple T-cellepitopes addressing the extensive polymorphism of HLA molecules(Paradiso et al., Vaccine Res., 1993, 2, 239-248), and universalT-helper epitopes compatible with human use. Exemplary T-helper epitopesinclude but are not limited to natural epitopes characterized fromtetanus toxoid (Valmor et al., J. Immunol., 1992, 149, 717-721) andnon-natural epitopes or engineered epitopes such as the pan HLADR-binding epitope PADRE (KXVAAWTLKAA (SEQ ID NO: 41); Immunity, 1994,1, 751-761).

Other types of carrier include but are not limited to biotin. The oligo-or polysaccharides conjugated to biotin or to a label are especiallydesigned for diagnosing S. flexneri infections.

Vaccine

The invention provides an immunogenic composition comprising aglycoconjugate as defined above, in a physiologically acceptablevehicle.

The vaccine composition includes one or more pharmaceutically acceptableexcipients or vehicles such as water, saline, glycerol, ethanol.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles.

The glycoconjugate of the present invention which induces protectiveantibodies against S. flexneri infection, in particular S. flexneri type2a are administered to a mammal subject, preferably a human, in anamount sufficient to prevent or attenuate the severity, extent ofduration of the infection by S. flexneri, in particular S. flexneri type2a.

Each vaccine dose comprises a therapeutically effective amount of oligo-or polysaccharide conjugate. Such amount will vary depending on thesubject being treated, the age and general condition of the subjectbeing treated, the capacity of the subject's immune response tosynthesize antibodies, the degree of protection desired, the severity ofthe condition to be treated, the particular oligo- or polysaccharideconjugate selected ant its mode of administration, among other factors.An appropriate effective amount can be readily determined by one ofskill in the art. A therapeutically effective amount will fall in arelatively broad range that can be determined through routine trials.

More particularly the oligo- or polysaccharide conjugate of theinvention will be administered in a therapeutically effective amountthat comprises from 1 to 1000 μg of oligo- or polysaccharide, preferably1 to 50 μg.

An optimal amount for a particular vaccine can be ascertained bystandard studies involving measuring the anti-LPS 2a antibody titers insubjects.

Following an initial vaccination, subjects may receive one or twobooster injections at about four week intervals.

According to a preferred embodiment of said immunogenic composition,said glycoconjugates comprises a pentasaccharide or a multimer thereofsuch as a decasaccharide or a pentadecasaccharide

The immunogenic composition of the invention may be administered with orwithout adjuvant. Adjuvants can be added directly to the vaccinecompositions or can be administered separately, either concurrently withor shortly after, administration of the vaccine. Such adjuvants includebut are not limited to aluminium salts (aluminium hydroxide),oil-in-water emulsion formulations with or without specific stimulatingagents such as muramyl peptides, saponin adjuvants, cytokines,detoxified mutants of bacteria toxins such as the cholera toxin, thepertussis toxin, or the E. coli heatlabile toxin.

The immunogenic composition of the invention may be administered withother immunogens or immunoregulatory agents, for example,immunoglobulins, cytokines, lymphokines and chemokines.

According to another preferred embodiment of said immunogeniccomposition, it comprises at least an immunogen which afford protectionagainst another pathogen, such as for example, S. flexneri serotype 1b,3a and 6, S. species such as S. dysenteriae 1 and S. sonnei or pathogensresponsible for diarrhoeal disease in humans [Vibrio cholerae (cholera),Salmonella typhimurium (typhoid), rotavirus, Enterotoxic strains of E.Coli (ETEC)].

Typically, the vaccine compositions are prepared as injectables eitheras liquid solutions or suspensions; or as solid forms suitable forsolution or suspension in liquid vehicle prior to injection. Thepreparation may be emulsified or encapsulated in liposomes for enhancedadjuvant effect.

Once formulated, the vaccine compositions may be administeredparenterally, by injection, either subcutaneous, intramuscular orintradermal.

Alternative formulations suitable for other mode of administrationinclude oral and intranasal formulations.

Antibodies

The invention provides monoclonal IgG antibodies immunoreactive with aserotype 2a-specific antigenic determinant of the O—SP of S. flexneritype 2a (O—SP or O—Ag) which are produced by an hybridoma cell linedeposited under the accession number I-3197, I-3198, I-3199, I-3200 andI-3201, on Apr. 20, 2004, at the Collection Nationale de Cultures deMicroorganismes, INSTITUT PASTEUR, 25 rue du Docteur Roux, 75724 PARISCEDEX 15, FRANCE.

The invention encompasses also the hybridoma cell line producing thehere above defined monoclonal IgG antibodies.

The monoclonal IgG antibodies according to the invention arerepresentative of the different IgG subclasses;

-   -   the hybridoma cell line I-3197 produces an IgG2a antibody        denominated hereafter A2-1,    -   the hybridoma cell line I-3198 produces an IgG3 antibody        denominated hereafter C1-7,    -   the hybridoma cell line I-3199 produces an IgG1 antibody        denominated hereafter D15-7,    -   the hybridoma cell line I-3200 produces an IgG2b antibody        denominated hereafter E4-1,

the hybridoma cell-line I-3201 produces an IgG1 antibody denominatedhereafter F22-4.

The invention provides also chimeric antibodies comprising: (i) afragment of the heavy and/or light chain(s) which is identical with orhomologous to the sequences of one of the here above defined mousemonoclonal IgG antibody, and (ii) the remainder of the heavy and orlight chain(s) which is identical with or homologous to the sequences ofan antibody from another species or belonging to another antibody classor subclass.

Accordingly, an advantageous embodiment of said chimeric antibody, is ahumanized antibody which contains minimal sequences from mouse origin.For the most part humanized antibodies are human immunoglobulins inwhich the residues from one or more CDR(s) are replaced by residues fromone or more CDR(s) of one of the here above defined mouse monoclonal IgGantibodies. Furthermore, humanized antibody may comprise residues whichare found neither in the human antibody, nor in the imported CDR(s) orframework (FR) sequences. These modifications are made to further refineand optimize antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains in which all or substantially all of the CDR regionscorrespond to those of the mouse monoclonal IgG antibody as here abovedefined, and all or substantially all of the FR regions are those of ahuman immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a domain of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

Preferably, said humanized antibody comprises the constant region froman IgG or an IgA, or at least the CH3 domains thereof.

More preferably, when said constant region is from an IgA. saidhumanized antibody comprises also a J chain so as to form dimeric IgAand/or a secretory component, so as to form secretory IgA.

According to another advantageous embodiment of said chimeric antibodyit comprises a Fab fragment from said mouse monoclonal IgG antibody anda constant region from a human IgA, or at least the CH3 domains thereof.

The invention provides also fragments from the here above definedmonoclonal IgG antibodies and deriving chimeric antibodies. Preferredfragments are functional fragments comprising the antigen recognitionand binding site such as: Fv or half of the Fv comprising only threeComplementarity-Determining-Regions (CDRs), Fab and Fab′₂.

Accordingly, an advantageous embodiment of said fragments is the CDRdefined by the sequences SEQ ID NO: 12 to 34.

The invention provides also the polynucleotides (DNA or RNA) encodingthe heavy and/or light chain from the here above defined antibodies, ora fragment thereof such as: a variable region (VL, VH) or a portionthereof such as a framework and/or CDR, and a constant region or aportion thereof such as a constant domain (CL, CH1, CH2, CH3).

The invention provides also the vectors comprising said polynucleotides.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors A vector according to the present invention comprises, but isnot limited to, a YAC (yeast artificial chromosome), a BAC (bacterialartificial), a baculovirus vector, a phage, a phagemid, a cosmid, aviral vector, a plasmid, a RNA vector or a linear or circular DNA or RNAmolecule which may consist of chromosomal, non chromosomal,semi-synthetic or synthetic DNA. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer generally to circular double stranded DNA loopswhich, in their vector form are not bound to the chromosome. Largenumbers of suitable vectors are known to those of skill in the art.

Preferably said vectors are expression vectors, wherein a sequenceencoding an antibody of the invention is placed under control ofappropriate transcriptional and translational control elements to permitproduction or synthesis of said protein. Therefore, said polynucleotideis comprised in an expression cassette. More particularly, the vectorcomprises a replication origin, a promoter operatively linked to saidencoding polynucleotide, a ribosome site, an RNA-splicing site (whengenomic DNA is used), a polyadenylation site and a transcriptiontermination site. It also can comprise an enhancer. Selection of thepromoter will depend upon the cell in which the polypeptide isexpressed.

The invention also concerns a prokaryotic or eukaryotic host cell thatis modified by a polynucleotide or a vector as defined above, preferablyan expression vector.

As used herein, a cell refers to a prokaryotic cell, such as a bacterialcell, or eukaryotic cell, such as an animal, plant or yeast cell.

The invention also concerns a non-human transgenic animal or atransgenic plant, wherein all or part of the cells are modified by apolynucleotide or a vector as defined above.

The polynucleotide sequence encoding the polypeptide of the inventionmay be prepared by any method known by the man skilled in the art. Forexample, it is amplified from a cDNA template, by polymerase chainreaction with specific primers. Preferably the codons of said cDNA arechosen to favour the expression of said protein in the desiredexpression system.

The recombinant vectors comprising said polynucleotide may be obtainedand introduced in a host cell by the well-known recombinant DNA andgenetic engineering techniques.

The antibody of the invention may be obtained by culturing the host cellcontaining an expression vector comprising a polynucleotide sequenceencoding said polypeptide, under conditions suitable for the expressionof the polypeptide, and recovering the polypeptide from the host cellculture.

Passive Protection

The invention provides a pharmaceutical composition comprising anantibody, as defined above or a functional fragment thereof; and aphysiologically acceptable vehicle.

The antibodies of the present invention which have a protective effectagainst S. flexneri infection, in particular S. flexneri type 2a areadministered to a mammal subject, preferably a human, in an amountsufficient to prevent or attenuate the severity, extent of duration ofthe infection by S. flexneri, in particular S. flexneri type 2a.

The administration of the antibody may be either prophylactic (prior tothe anticipated exposure to S. flexneri) or therapeutical (after theinitiation of the infection, at or shortly after the onset of thesymptoms).

The dosage of the antibodies will vary depending upon factors as thesubject's age, weight and species. In general, the dosage of theantibody is in the range of from about 1 mg/kg to 10 mg/kg body weight.

Preferably, said antibody is a humanized antibody of the IgG or the IgAclass.

The route of administration of the antibody may be oral or systemic, forexample, subcutaneous, intramuscular or intravenous.

Diagnosis

The antibodies and the oligo- or polysaccharides according to thepresent invention are used, in vitro, as S. flexneri type 2a specificdiagnostic reagents in standard immunoassays.

The antibodies according to the present invention are used to test forthe presence of S. flexneri type 2a in biological samples, forestablishing the diagnosis of shigellosis in an individual presenting adiarrhoeal disease.

Alternatively, the oligo- or polysaccharides according to the presentinvention are used to test the presence of S. flexneri type 2a-specificantibodies. Oligo- or polysaccharides may be used for epidemiologicalstudies, for example for determining the geographic distribution and/orthe evolution of S. flexneri type 2a infection worldwide, as well as forevaluating the S. flexneri type 2a-specific antibody response induced byan immunogen.

The antibodies and the oligo- or polysaccharides according to thepresent invention may be advantageously labelled and/or immobilized ontoa solid phase, according to standard protocols known to the man skilledin the art. Such labels include, but are not limited to, enzymes(alkaline phosphatase, peroxydase), luminescent or fluorescentmolecules. For example an oligo- or polysaccharide conjugated tobiotine, according to the present invention may be immobilized onto asolid phase, to detect the presence of S. flexneri type 2a-specificantibodies in biological samples.

Such immunoassays include, but are not limited to, agglutination assays,radioimmunoassay, enzyme-linked immunosorbent assays, fluorescenceassays, western-blots and the like.

Such assays may be for example, of direct format (where the labelledantibody/oligo- or polysaccharide is reactive with the antigen/antibodyto be detected), an indirect format (where a labelled secondary antibodyis reactive with said antibody/oligo- or polysaccharide), a competitiveformat (addition of a labelled antibody/oligo- or polysaccharide), or asandwich format (where both labelled and unlabelled antibodies areused).

For all therapeutic, prophylactic and diagnostic uses, the oligo- orpolysaccharides of the invention, alone or linked to a carrier, as wellas antibodies and other necessary reagents and appropriate devices andaccessories may be provided in kit form so as to be readily availableand easily used.

Detailed Description of the Preparation of the Molecules

The instant invention is based on the characterization of the antigenicdeterminants of S. flexneri 2a O—SP recognized by serotype-specificprotective monoclonal antibodies. The synthesis, as their methylglycosides, of a panel of oligosaccharides representative of fragmentsof S. flexneri 2a O—SP was thus undertaken to be used as probes in thestudy of antibody recognition.

         A              B               E               C              D2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-[α-D-Glcp-(1→4)]-α-L-Rhap-(1→3)-β-D-GlcNAcp(1→

The O—SP of S. flexneri 2a is a heteropolysaccharide defined by thepentasaccharide repeating unit I. {(D. A. R. Simmons, Bacteriol. Reviews1971, 35, 117-148; A. A. Lindberg, A. Karnell, A. Weintraub, Rev.Infect. Dis. 1991, 13, S279-S284) It features a linear tetrasaccharidebackbone, which is common to all S. flexneri O-antigens, except serotype6, and comprises a N-acetyl glucosamine and three rhamnose residues,together with an α-D-glucopyranose residue branched at position 4 ofrhamnose C. We have already reported on the synthesis of the methylglycosides of various fragments of the O—SP, including the known ECdisaccharide, (J. M. Berry, G. G. S. Dutton, Can. J. Chem. 1974, 54,681-683; G. M. Lipkind, A. S. Shashkov, A. V. Nikolaev, S. S. Mamyan, N.K. Kochetkov, Bioorg. Khim. 1987, 13, 1081-1092; L. A. Mulard, C.Costachel, P. J. Sansonetti, J. Carbohydr. Chem. 2000, 19, 849-877) theECD (L. A. Mulard, C. Costachel, P. J. Sansonetti, J. Carbohydr. Chem.2000, 19, 849-877) and B(E)C (L. A. Mulard, C. Costachel, P. J.Sansonetti, J. Carbohydr. Chem. 2000, 19, 849-877) trisaccharides, theECDA (F. Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002, 13,2211-2222) and AB(E)C (C. Costachel, P. J. Sansonetti, L. A. Mulard, J.Carbohydr. Chem. 2000, 19, 1131-1150) tetrasaccharides, the B(E)CDA (F.Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002, 13, 2211-2222) andDAB(E)C (C. Costachel, P. J. Sansonetti, L. A. Mulard, J. Carbohydr.Chem. 2000, 19, 1131-1150) pentasaccharides and more recently theB(E)CDAB(E)C octasaccharide (F. Bélot, C. Costachel, K. Wright, A.Phalipon, L. A. Mulard, Tetrahedron. Lett. 2002, 43, 8215-8218).

In the following, we report on the synthesis of the ECDAB, AB(E)CDpentasaccharides as well on that of the B(E)CD tetrasaccharide as theirmethyl glycosides, 1, 2 and 3, respectively. We also report on thesynthesis of a pentasaccharide DAB(E)C building block (201) and that ofthe corresponding trichloroacetimidate donor 203. The decasaccharideD′A′B′(E′)C′DAB(E)C fragment, was prepared as its methyl glycoside(301).

I—Synthesis of Oligo- and Polysaccharides According to the Invention

A—Synthesis of a Tetra- and Two Pentasaccharide Fragments of theO-Specific Polysaccharide of Shigella flexneri Serotype 2a:

The synthesis of the methyl glycosides of the ECDAB, AB(E)CDpentasaccharides and that of the B(E)CD tetrasaccharide, 101, 102 and103, respectively, is reported in the following.

Analysis of the targets shows that all the glycosylation reactions toset up involve 1,2-trans glycosidic linkages except for that at the E-Cjunction which is 1,2-cis. Consequently, the syntheses described hereinrely on key EC disaccharide building blocks as well as on appropriate A,B and D monosaccharide synthons.

Synthesis of the linear ECDAB-OMe pentasaccharide (101): Based onearlier findings in the series which have demonstrated that the C-Dlinkage was an appropriate disconnection site. (F. Segat, L. A. Mulard,Tetrahedron: Asymmetry 2002, 13, 2211-2222; F. Bélot, C. Costachel, K.Wright, A. Phalipon, L. A. Mulard, Tetrahedron. Lett. 2002, 43,8215-8218; F. Bélot, K. Wright, C. Costachel, A. Phalipon, L. A. Mulard,J. Org. Chem. 2004, 69, 1060-1074) Consequently, the synthesis of 101was designed (FIG. 1) based on the glycosylation of the known ECtrichloroacetimidate donor 114, (L. A. Mulard, C. Costachel, P. J.Sansonetti, J. Carbohydr. Chem. 2000, 19, 849-877) obtained in threesteps (69%) from the key diol 113, (F. Segat, L. A. Mulard, Tetrahedron:Asymmetry 2002, 13, 2211-2222) and the DAB trisaccharide acceptor 112.The latter was obtained by the stepwise condensation of knownmonosaccharide precursors, readily available by selective protection,deprotection and activation sequences. Thus, TMSOTf-catalysedcondensation of the rhamnopyranoside acceptor 104 (V. Pozsgay, J.-R.Brisson, H. J. Jennings, Can. J. Chem. 1987, 65, 2764-2769) with thetrichloroacetimidate donor 5 (J. C. Castro-Palomino, M. H. Rensoli, V.V. Bencomo, J. Carbohydr. Chem. 1996, 15, 137-146) in diethyl ether togive the fully protected rhamnobioside 106, and subsequentde-O-acetylation gave the AB disaccharide acceptor 107 in 91% overallyield, which compares favourably with the previously describedpreparation using the corresponding 1-O-acetyl donor. (V. Pozsgay, J.-R.Brisson, H. J. Jennings, Can. J. Chem. 1987, 65, 2764-2769) Analogouslyto previous work in a related series, (F. Bélot, K. Wright, C.Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074)the known glucosaminyl trichloroacetimidate donor 109, (J. C.Castro-Palomino, R. R. Schmidt, Tetrahedron Lett. 1995, 36, 5343-5346)was chosen as the precursor to residue D. Conventional glycosylation of107 with 109 was best performed in acetonitrile using tintrifluoromethanesulfonate (Sn(OTf)₂) as the catalyst (A. Lubineau, A.Malleron, Tetrahadron Lett. 1985, 26, 1713-1716) to give the fullyprotected trisaccharide 110 in 72% yield (extracted from the ¹H NMRspectrum). When TMSOTf was used instead of Sn(OTf)₂, 110 was formed inlower yield (52%) outlining the sensitivity of the tetrachlorophtaloylgroup to these stronger conditions, as previously noted. (L. Lay, L.Manzoni, R. R. Schmidt, Carbohydr. Res. 1998, 310, 157-171) A three stepprocess including heating 110 with ethylenediamine in dry ethanol, (J.S. Debenham, R. Madsen, C. Roberts, B. Fraser-Reid, J. Am. Chem. Soc.1995, 117, 3302-3303) ensuing N-acetylation with acetic anhydride, andde-O-acetylation under Zemplén conditions, furnished the triol 111 (51%from 107). It was next protected at positions 4_(D) and 6_(D) byregioselective introduction of an isopropylidene acetal upon reactionwith 2,2-dimethoxypropane under acid-catalysis to give 112 (96%). Thelatter acetal-protecting group was selected based on data previouslyobtained when synthesizing shorter fragments in the series which hadoutlined the interest of using 4,6-O-isopropylidene-glucosaminylintermediates instead of the more common benzylidene analogues. (L. A.Mulard, C. Costachel, P. J. Sansonetti, J. Carbohydr. Chem. 2000, 19,849-877) Once the two key building blocks were made available, theircondensation was performed in dichloromethane in the presence of acatalytic amount of TMSOTf to give the fully protected pentasaccharide115 (84%). Conventional stepwise deprotection involving (i) acidichydrolysis of the isopropylidene acetal using 90% aq TFA to give diol116 (95%), (ii) conversion of the latter into the corresponding tetraol117 under Zemplén conditions (86%), and (iii) final hydrogenolysis ofthe benzyl protecting groups, gave the linear pentasaccharide target 101in 81% yield.

Synthesis of the AB(E)CD pentasaccharide 102 and of the B(E)CDtetrasaccharide 103. For reasons mentioned above, the glucosaminylacceptor 118, (L. A. Mulard, C. Costachel, P. J. Sansonetti, J.Carbohydr. Chem. 2000, 19, 849-877) protected at its 4 and 6 hydroxylgroups by an isopropylidene acetal was the precursor of choice forresidue D (FIG. 2). In the past, introduction of residue B at position3_(C) was performed on a 2_(C)-O-benzoylated EC acceptor resulting fromthe regioselective acidic hydrolysis of the corresponding 2,3-orthoesterintermediate. (F. Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002, 13,2211-2222; C. Costachel, P. J. Sansonetti, L. A. Mulard, J. Carbohydr.Chem. 2000, 19, 1131-1150) It rapidly occurred to us that opening of theintermediate phenyl orthoester was not compatible with the presence of4_(D),6_(D)-O-isopropylidene acetal. For that reason, thetrichloroacetimidate donor 119, suitably benzoylated at position 2_(C)and orthogonally protected by a chloroacetyl group at position 3_(C) wasused as the EC building block instead of the previously used 114.Protection at the 2-OH of the rhamnosyl precursor to residue B was alsocrucial in the synthesis of 102. Indeed, most of our previous work inthe series relied on the use of the known 2-O-acetyl rhamnopyranosyldonor 105, In the reported syntheses, (C. Costachel, P. J. Sansonetti,L. A. Mulard, J. Carbohydr. Chem. 2000, 19, 1131-1150) selectivede-O-acetylation at position 2_(B) in the presence of a 2_(C)-O-benzoatewas best performed by treatment with methanolic HBF₄.OEt₂ for five daysClearly, such de-O-acetylation conditions are not compatible with thepresence of an isopropylidene acetal on the molecule. To overcome thislimitation, the corresponding 2-O-chloroacetyl rhamnopyranosyltrichloroacetimidate 120 was selected as an alternate donor. In theory,the latter could also serve as an appropriate precursor to residue A.

Regioselective conversion of diol 113 into its 2-O-benzoylatedcounterpart 121 was performed as described (FIG. 3). (F. Segat, L. A.Mulard, Tetrahedron: Asymmetry 2002, 13, 2211-2222) Treatment of thelatter with chloroacetic anhydride and pyridine gave the orthogonallyprotected 122 (95%), which was smoothly de-O-allylated to yield thecorresponding hemiacetal 123 (91%) by a two-step process, involving (i)iridium (I)-promoted isomerisation (J. J. Oltvoort, C. A. A. vanBoeckel, J. H. der Koning, J. van Boom, Synthesis 1981 305-308) of theallyl glycoside and (ii) subsequent hydrolysis in the presence ofiodine. (M. A. Nashed, L. Anderson, J. Chem. Soc. Chem. Commun. 19821274-1282) The selected trichloroacetimidate leaving group wassuccessfully introduced by treatment of 123 with trichloroacetonitrilein the presence of DBU, which resulted in the formation of 119 (84%)together with the recovery of some starting hemiacetal (14%) sincepartial hydrolysis during column chromatography could not be avoided.TMSOTf-mediated glycosylation of donor 119 and acceptor 118 furnishedthe fully protected ECD trisaccharide (124, 80%), which was readilyconverted to the required acceptor 125 upon selective deblocking of thechloroacetyl protecting group with thiourea (97%). Following thetwo-step protocol described above for the preparation of 119, the knownallyl rhamnopyranoside 127, (P. Westerduin, P. E. der Haan, M. J. Dees,J. H. van Boom, Carbohydr. Res. 1988, 180, 195-205) bearing a2-O-chloroacetyl protecting group, was converted to the hemiacetal 128(85%) (FIG. 4). Next, treatment of the latter with trichloroacetonitrileand a slight amount of DBU gave at best donor 120 in a yield of 73%.Although the isolated yield of 120 was not better (72%), running theactivation step in the presence of K₂CO₃ instead of DBU resulted in amore reproducible isolated yield of the activated donor. Glycosylationof the ECD acceptor 125 and the B donor 120 was attempted under variousconditions of solvent and catalyst. Whatever the conditions, hardlyseparable mixtures of compounds were obtained, among which the yield ofthe target tetrasaccharide reached 45-50%. Running the condensation inEt₂O in the presence of TMSOTf as the promoter were the best conditionstested, although the expected tetrasaccharide 129 was often slightlycontaminated with glycosylation intermediates such as the silylated 126or the orthoester 135 (FIG. 5), as suggested from mass spectroscopyanalysis and NMR data. In fact, the nature of the latter was fullyascertained at the next step in the synthesis. Indeed, full recovery ofthe starting material was observed upon treatment of 135 with thiourea.On the contrary, treatment of a mixture of the condensation products 129and supposedly 126 under the same conditions led to the expectedtetrasaccharide acceptor 131 and the trisaccharide acceptor 125 (notdescribed). The βB-tetrasaccharide isomer could not be detected at thisstage, indicating that the corresponding chloroacetylated βB-anomer wasprobably not part of the initial mixture. Formation of the starting 125during the dechloroacetylation step was not unexpected, since loss of atrimethylsilyl group under similar treatment was observed for a modelcompound (not described). Although the fluoride analog corresponding todonor 120 has been used successfully in a prior report, (P. Westerduin,P. E. der Haan, M. J. Dees, J. H. van Boom, Carbohydr. Res. 1988, 180,195-205) the poor yield of 129 may be, in part, associated to thesensitivity of the chloroacetyl group to the glycosylation conditions.Thus, in order to investigate the poor outcome of the condensationreaction, the donor properties of the chloroacetylated 120 were comparedto that of the more common acetylated 105. When methyl rhamnopyranoside104 was condensed with 120 as described for the preparation of 106, therhamnobioside 108 was isolated in 67% yield. This result tends tosuggest that indeed the acetylated 5 is a more powerful donor than 120.

Starting from 120 and 125, the isolated yield of the tetrasaccharideacceptor 131 was 34%, which encouraged us to reconsider the use of 105as a precursor to residues B and A in the synthesis of 102. Condensationof 105 and 125 in CH₂Cl₂ using TMSOTf as the promoter furnished thecorresponding tetrasaccharide 130 (72%). However, even though the yieldof 131 was better than that of 129, slight contamination by thesilylated side-product 126 was again apparent, outlining the somewhatpoor reactivity of the ECD acceptor. Subsequent treatment of 130 with a0.4 M ethanolic solution of guanidine (N. Kunesh, C. Miet, J. Poisson,Tetrahadron Lett. 1987, 28, 3569-3572) resulted in selective2_(B)-O-deacetylation to give 131 in a satisfactory 83% yield, whichoutlined the interest of the method. However, previous experience inother closely related series has shown that the selectivity of themethod was highly dependent on the nature of the substrate. Clearly, the2-O-acetylated donor 105 was preferred to the chloroacetate analogue120. Condensation of the tetrasaccharide acceptor 131 and donor 105 inthe presence of TMSOTf gave the fully protected pentasaccharide 132 in ayield of 52%. TFA-mediated hydrolysis of the isopropylidene acetalfollowed by transesterification of the ester groups and subsequentconventional hydrogenolysis of the benzyl ethers finally gave the targetpentasaccharide 2 (88%).

Alternatively, the fully protected tetrasaccharide 130 was converted thediol 136 by acidic removal of the isopropylidene acetal (85%), andsubsequently to the corresponding tetraol 137 upon transesterification(83%). Final hydrogenolysis of the benzyl groups furnished the targettetrasaccharide 103 (71%) (FIG. 6).

Noteworthy, in the case of intermediates 133 and 136, removal of theesters required heating of the reaction mixtures, whereas de-O-acylationof 117 proceeded smoothly at rt. Occurring most probably as aconsequence to the branched nature of compounds 133 and 136, sterichindrance and isolation of the acyl groups (Z. Szurmai, A. Liptak, G.Snatzke, Carbohydr. Res. 1990, 200, 201-208) may best explain thephenomenon. Steric hindrance may also account for the poor outcome ofthe condensation of the ECD acceptor 125 with the B donors 120 and 105.Interestingly, ¹³C NMR data support this hypothesis. Although no alteredsignals could be seen in the ¹³C NMR spectrum of the ECD acceptor 125 orin the ¹³C NMR spectra of the fully protected precursor 124, significantdisturbance of several signals in the ¹³C NMR spectra of the tetra- andpentasaccharides were seen repeatedly. At the protected and partiallyprotected stage, major altered signals are those tentatively assigned toC-3_(C) and C-4_(C). Besides, signals assigned to C-2_(D), C-3_(D) aswell as to C-1_(B) are significantly broader than expected. Loss ofconformational flexibility at the C ring is not totally unexpectedespecially since the carbons involved are those corresponding to thebranching points. Of particular interest however, was the observationthat residue D, the N-acetyl-glucosaminyl residue, was also partiallyconstrained. Full conformational freedom of residue D is recovered whenthe B(E)CD and AB(E)CD oligosaccharides are in their free form. However,this observation does not stand true for residue C since characteristicbroad signals for C-3_(C) and C-4_(C) as well for C-1_(B) and C-1_(E)are still present in the ¹³C NMR spectra of compounds 102 and 103,respectively. Overall, these observations suggest a somewhat compactorganisation at the branching point of the B(E)CD structure. It is worthmentioning that none of these disturbed signals are seen in the ¹³C NMRspectra of the oligosaccharides corresponding to the linear ECDABfragment.

The synthesis of the methyl glycoside (102) of the repeating unit I ofthe S. flexneri 2a O—SP, together with that of the correspondingframe-shifted pentasaccharide 101 and tetrasaccharide 103 weredescribed. All the methyl glycosides of the di- to pentasaccharidesobtained by circular permutation of the monosaccharide residuespartaking in the linear backbone of I, and comprising the EC portion,are now available in the laboratory. Their binding to a set ofprotective monoclonal IgG antibodies will be reported elsewhere.

B—Synthesis of a Pentasaccharide Building Block of the O-SpecificPolysaccharide of Shigella flexneri serotype 2a: DAB(E)C

In the following, a synthesis of the DAB(E)C pentasaccharide 201, whichis protected in an orthogonal fashion at position O-3_(D) with an acetylgroup and at the reducing end by an allyl group. At this stage, theacetamido function is already present at position 2_(D). Compound 201may be converted to the corresponding alcohol 202, which acts as andonor and a masked donor, or to the trichloroacetimidate 203 which actsas an acceptor allowing subsequent chain elongation at the non-reducingend (FIG. 7). Previous work in the laboratory has shown that in order toconstruct the DAB(E)C sequence, the linear approach involving stepwiseelongation at the non-reducing end, was more suitable than the blockwiseone.

D-glucosamine unit (D). In order to limit the number of steps at thepentasaccharide level, we reasoned that an appropriate precursor toresidue D should have (i) permanent protecting groups at positions 4 and6, (ii) a participating group at position 2 and (iii) an orthogonalprotecting group at position 3, allowing easy cleavage. As they allow awide range of protecting group manipulations previously to ultimateactivation, thioglycosides are highly convenient masked donors.Recently, two sets of non-malodorous thioglycosyl donors have beenproposed (H. Dohi, Y. Nishida, T. Takeda, K. Kobayashi, Carbohydr. Res.2002, 337, 983-989; H. Matsui, J.-I. Furukawa, T. Awano, N. Nishi, N.Sakairi, Chem. Lett. 2000, 29, 326-327), among which the thiododecanylmoiety was selected (FIG. 8). Thus, the known peracetylatedtrichloroacetamide 204 (G. Blatter, J.-M. Beau, J.-C. Jacquinet,Carbohydr. Res. 1994, 260, 189-202) was reacted with dodecanthiol in thepresence of BF₃.OEt₂ to give thioglycoside 205 in high yield (97%).Zemplén deacetylation cleanly afforded the corresponding triol 206,which was selectively protected at position 4 and 6 upon reaction with2,2-dimethoxypropane to give 207 (80% from 204). Indeed, previousobservations in the series have demonstrated that4,6-O-isopropylidene-D-glucosaminyl derivatives were highly suitableprecursors to residue D. (L. A. Mulard, C. Costachel, P. J. Sansonetti,J. Carbohydr. Chem. 2000, 19, 849-877; F. Bélot, C. Costachel, K.Wright, A. Phalipon, L. A. Mulard, Tetrahedron. Lett. 2002, 43,8215-8218) Next, conventional acetylation of 207 gave the requiredthioglycoside donor 208.

L-Rhamnose units (A, B): Previous work in the series was mostly based onthe use of the 2-O-acetyl trichloroacetimidate rhamnopyranosyl donor213. (C. Costachel, P. J. Sansonetti, L. A. Mulard, J. Carbohydr. Chem.2000, 19, 1131-1150; F. Bélot, K. Wright, C. Costachel, A. Phalipon, L.A. Mulard, J. Org. Chem. 2004, 69, 1060-1074) Condensation yields wereexcellent. However, the acetyl protecting group is not fully orthogonalto the benzoyl one, which is a weak point in the strategy sinceselective de-O-acetylation is required twice. The levulinate on thecontrary is fully orthogonal to either benzyl or allyl ethers, and tobenzoates. The 2-O-levulinoyl trichloroacetimidate donor 212 was thusevaluated as an alternative to 213. It was prepared from the known allylrhamnopyranoside 209 (P. Westerduin, P. E. der Haan, M. J. Dees, J. H.van Boom, Carbohydr. Res. 1988, 180, 195-205) in three steps (FIG. 9).Indeed, treatment of 209 with levulinic acid gave the fully protected210 (95%), deallylation of which proceeded in two steps based on (i)isomerisation of the allyl group into the prop-1-enyl ether using aniridium complex, (J. J. Oltvoort, C. A. A. van Boeckel, J. H. derKoning, J. van Boom, Synthesis 1981 305-308) and (ii) subsequentoxidative cleavage of the latter to give the hemiacetal 211 (85-95%).(M. A. Nashed, L. Anderson, J. Chem. Soc. Chem. Commun. 1982 1274-1282)Reaction of the latter with trichloroacetonitrile in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) resulted in the required donor212 (95%).

Synthesis of the pentasaccharide 201 (FIG. 10): The known allylglycoside 214, acting as an EC acceptor, temporarily protected at theanomeric position and having a participating group at position 2_(C),was prepared as described in 63% yield from allyl2,3-O-isopropylidene-α-L-rhamnopyranoside. (F. Segat, L. A. Mulard,Tetrahedron: Asymmetry 2002, 13, 2211-2222) Its condensation with thetrichloroacetimidate donor 212, performed in the presence of a catalyticamount of TMSOTf, afforded the fully protected trisaccharide 215(80-95%), and subsequently the known B(E)C acceptor 216 (F. Bélot, K.Wright, C. Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69,1060-1074) upon selective removal of the O-levulinoyl group withhydrazine hydrate (80-94%). Starting from 216, this two-step process wasrepeated to give first the fully protected 217 (54-90%), then the knownAB(E)C acceptor 218 (F. Bélot, K. Wright, C. Costachel, A. Phalipon, L.A. Mulard, J. Org. Chem. 2004, 69, 1060-1074) in 80-94% yield.Considering that selective deblocking at positions 2_(B) and 2_(A) wascompleted in overnight runs instead of the 5 days required for eachcorresponding chemoselective O-deacetylation steps, the use of the2-O-levulinoyl donor 212 appeared as a suitable alternative to that of213. Using a mixture of NIS and triflic acid as the promoter,condensation of the tetrasaccharide acceptor 218 with the thioglycosidedonor 208 gave the key intermediate 219 in 58% yield. Althoughalternative conditions in terms of promoters and solvents (notdescribed) were tested, this rather low yield could not be improved.Bu₃SnH mediated radical dechlorination of 219 in the presence of acatalytic amount of AIBN readily afforded the corresponding acetamidokey intermediate 201 (74%). On one hand, compound 201 may be efficientlyconverted to the acceptor building block 202 under Zemplén conditions.On the other hand, it was smoothly deallylated into the hemiacetal 220,following a two-step process as described above. Next, treatment of 220with trichloroacetonitrile and DBU allowed its conversion to thebuilding block 3 (82% from 201).

C—Convergent Synthesis of the Decasaccharide D′A′B′(E′)C′DAB(E)C

Considering its dimeric nature, a convergent synthetic strategy to thetarget methyl glycoside of the decasaccharide D′A′B′(E′)C′DAB(E)C (301)was considered. Indeed, retrosynthetic analysis, supported by previouswork in the field, (Bélot, F.; Costachel, C.; Wright, K.; Phalipon, A.;Mulard, L. A. Tetrahedron. Lett. 2002, 43, 8215-8218; Kochetkov, N. K.;Byramova, N. E.; Tsvetkov, Y. E.; Backinovsky, L. V. Tetrahedron 1985,41, 3363-3375; Pinto, B. M.; Reimer, K. B.; Morissette, D. G.; Bundle,D. R. J. Org. Chem. 1989, 54, 2650-2656; Pinto, B. M.; Reimer, K. B.;Morissette, D. G.; Bundle, D. R. J. Chem. Soc. Perkin Trans. 1 1990,293-299) indicated that disconnections at the C-D linkage, thus based ontwo DAB(E)C branched pentasaccharides corresponding to a frame-shiftedrepeating unit I, would be the most advantageous (FIG. 11). Such astrategy would involve a pentasaccharide acceptor easily derived fromthe known methyl glycoside 302 (Costachel, C.; Sansonetti, P. J.;Mulard, L. A. J. Carbohydr. Chem. 2000, 19, 1131-1150) or from thecorresponding N-acetylated analogue 303 and a pentasaccharide donorbearing a 2-O-acyl protecting group at the reducing residue (C) in orderto direct glycosylation towards the desired stereochemistry. Dependingon the nature of the 2-N-acyl group in residue D, the latter couldderive from the allyl glycosides 304 or 305. Besides, bearing in mindthat the major drawbacks of the linear synthesis of pentasaccharide 302reported so far (Costachel, C.; Sansonetti, P. J.; Mulard, L. A. J.Carbohydr. Chem. 2000, 19, 1131-1150) dealt with the selectivedeblocking of key hydroxyl groups to allow further chain elongation, wedescribe herein various attempts at a convergent synthesis of the fullyprotected DAB(E)C pentasaccharide as its methyl (302, 303) or allyl(304, 305) glycosides. Precedents concerning a related serotype of S.flexneri have indicated that disconnection at the D-A linkage should beavoided (Pinto, B. M.; Reimer, K. B.; Morissette, D. G.; Bundle, D. R.J. Org. Chem. 1989, 54, 2650-2656; Pinto, B. M.; Reimer, K. B.;Morissette, D. G.; Bundle, D. R. J. Chem. Soc. Perkin Trans. 1 1990,293-299). To our knowledge, disconnection at the B-C linkage was neverattempted in the series. However, disconnection at the A-B linkage,based on the use of a combination of a bromide disaccharide donor andHg(CN)₂/HgBr₂ as the promoter, was reported once. (N. K. Kochetkov, N.E. Byramova, Y. E. Tsvetkov, L. V. Backinovsky, Tetrahedron 1985, 41,3363-3375) In the latter case concerning the synthesis of the linearDABC tetrasaccharide, the condensation of two disaccharide buildingblocks was found more effective than the stepwise strategy. Both routeswere considered in the following study. The nature of the repeating unitI indicated that any blockwise synthesis involving such linkages wouldrely on donors lacking any participating group at position 2 of thereducing residue, thus the relevance of this strategy may be questioned.Nevertheless, although β-glycoside formation was observed occasionally,(Srivastava, O. P.; Hindsgaul, O. Can. J. Chem. 1986, 64, 2324-2330) thegood α-stereoselectivity reported on several occasions in the literaturefor glycosylation reactions based on mannobiosyl donors (Ogawa, T.;Kitajma, T.; Nukada, T. Carbohydr. Res. 1983, 123, c5-c7; Ogawa, T.;Sugimoto, M.; Kitajma, T.; Sadozai, K. K.; Nukuda, T. Tetrahadron Lett.1986, 27, 5639-5742) and derivatives such as perosaminyl analogues (LeiP. S; Ogawa, Y; Kovac, P. Carbohydr. Res. 1996, 281, 47-60; Kihlberg,J.; Eichler, E.; Bundle, D. R. Carbohydr. Res. 1991, 211, 59-75; Peters,T.; Bundle, D. R. Can. J. Chem. 1989, 67, 491-496) or rhamnopyranosyldonors that were either glycosylated at C-2 (Reimer, K. B.; Harris, S.L.; Varma, V.; Pinto, B. M. Carbohydr. Res. 1992, 228, 399-414), orblocked at this position with a non participating group (Varga, Z.;Bajza, I.; Batta, G.; Liptak, A. Tetrahedron Lett. 2001, 42, 5283-5286),encouraged the evaluation of the above mentioned block strategies. Tofollow up the work developed thus far in the S. flexneri 2a series,emphasis was placed on the use of the use of trichloroacetimidate (TCA)chemistry (Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.1994, 50, 21-123).

Strategy based on the disconnection at the A-B linkage (FIG. 11, routea): Such a strategy involves the coupling of suitable DA donors to anappropriate B(E)C acceptor. Taking into account the glycosylationchemistry, two sets of disaccharide building blocks (306, 307, 308),easily obtained from known monosaccharide precursors which were readilyavailable by standard protecting group/activation strategies, wereselected (FIG. 11). Thus, condensation of the allyl rhamnopyranoside314, (Westerduin, P.; der Haan, P. E.; Des, M. J.; van Boom, J. H.Carbohydr. Res. 1988, 180, 195-205) as precursor to residue A, with theglucosaminyl trichloroacetimidate 316, (Blatter, G.; Beau, J.-M.;Jacquinet, J.-C. Carbohydr. Res. 1994, 260, 189-202) as precursor toresidue D, was performed in the presence of a catalytic amount of TMSOTfto give the fully protected disaccharide 317 (99%). Selectivedeallylation of 317 proceeded in two steps involving (i)iridium(I)-catalysed isomerisation of the allyl glycoside into thecorresponding 1-O-propenyl glycoside (Oltvoort, J. J.; van Boeckel, C.A. A.; der Koning, J. H. d.; van Boom, J. Synthesis 1981, 305-308) and(ii) hydrolysis of the latter (Gigg, R.; Warren, C. D. J. Chem. Soc. C1968, 1903-1911; Gigg, R.; Payne, S.; Conant, R. J. Carbohydr. Chem.1983, 2, 207-223). The resulting hemiacetal 318 (81%) was converted intothe trichloroacetimidate 306 (78%) by treatment withtrichloroacetonitrile in the presence of a catalytic amount of DBU (FIG.12). Knowing from previous experience that conversion of thetrichloroacetamide moiety at position 2 of residue D(2_(D)-N-trichloroacetyl) into the required 2_(D)-N-acetyl group couldbe somewhat low-yielding, we took advantage of the blockwise approach toperform the above-mentioned transformation at an early stage in thesynthesis. Thus, the disaccharide intermediate 317 was converted to thecorresponding 319 (90%) upon overnight treatment with a saturatedammonia methanolic solution and subsequent peracetylation. Conversion of319 into the hemiacetal 320 (69%), and next into the requiredtrichloroacetimidate donor 307 (86%), followed the procedure describedabove for the preparation of 306 from 317. Where glycosylation isconcerned, the bifunctional role of thioglycosides as protectedacceptors and masked donors is highly appreciated. (S. Oscarson,Carbohydrates in chemistry and biology. Part 1: Chemistry of saccharides2000, 2, 93) Thus, the thiophenyl disaccharide 308 was considered as apossible alternative to the use of the more reactivetrichloroacetimidates 306 and 307. It was synthesized in 97% yield bycondensing the known thiophenyl rhamnopyranoside 315 (Lau, R.; Schuele,G.; Schwaneberg, U.; Ziegler, T. Liebigs Ann. Org. Bioorg. Chem. 1995,10, 1745-1754) and 316 in the presence of a catalytic amount of TMSOTf(FIG. 12). To fulfil the requirements of the synthesis of 301, twodifferent trisaccharide building blocks were used, namely either theknown methyl glycoside 309 (Costachel, C.; Sansonetti, P. J.; Mulard, L.A. J. Carbohydr. Chem. 2000, 19, 1131-1150) or the corresponding allylglycoside 310, obtained from the known 2_(B)-O-acetylated trisaccharide342 (see below and FIG. 15) (Segat, F.; Mulard, L. A. Tetrahedron:Asymmetry 2002, 13, 2211-2222). Condensation of the trisaccharideacceptor 309 and the trichloroacetimidate donor 306 was attempted undervarious conditions of solvent, temperature and promoter. The α-linkedcondensation product, i.e. the known pentasaccharide 302, (Costachel,C.; Sansonetti, P. J.; Mulard, L. A. J. Carbohydr. Chem. 2000, 19,1131-1150) was at best isolated in 41% yield providing that theglycosylation reaction was run in acetonitrile in the presence of acatalytic amount of TMSOTf, following the inverted procedure protocol(Schmidt, R. R.; Toepfer, A. Tetrahedron Lett. 1991, 32, 3353-3356;Bommer, R.; Kinzy, W.; Schmidt, R. R. Liebigs Ann. Chem. 1991, 425-433)to minimize degradation of the donor. Although the α-selectivity of theglycosylation reaction was good, yields of pentasaccharide remained low,and, as anticipated, use of the alternate trichloroacetimidate donor 307to give 303 did not result in any improvement (not described).Rearrangement of the activated donor into the corresponding inerttrichloroacetamide was observed previously in glycosylation reactionsbased on trichloroacetimidate donors lacking a participating group atposition 2 of the reducing residue. (K. H. Sadozai, T. Nukada, Y. Ito,Y. Nakahara, T. Ogawa, Carbohydr. Res. 1986, 157, 101-123) Although theexpected side-product was not isolated in any of the attemptedglycosylation with 306 or 307, it was anticipated that the use of analternate glycosylation chemistry would prevent such side-reaction, andpossibly favour the condensation. However, reaction of thiophenyl donor308 and acceptor 310 in the presence of N-iodosuccinimide and catalytictriflic acid did not prove any better as it resulted in mixtures ofproducts from which the target 304 was isolated in very low yield, 10%at best. This strategy was thus not considered any further.

Strategy based on the disconnection at the B-C linkage (FIG. 11, routeb). It was hypothesized that the good α-selectivity, but poor yields, ofthe condensation of the various DA donors with the B(E)C acceptors 309and 310 might result from the poor nucleophilicity of the axial hydroxylat position 2_(B). Thus, we next turned to the 3_(C)-OH as a possibleelongation site in the design of a block synthesis of pentasaccharide305. Considering such a disconnection approach suggests the use of a DABtrisaccharide donor for coupling to an EC disaccharide acceptor. As thetarget pentasaccharide should serve as an appropriate donor in theconstruction of 301, we reasoned that an acyl participating group had tobe present at its position 2_(C). Thus, two 2_(C)-O-acylated EC buildingblocks, 311 or 312, were considered. In order to avoid any unnecessarydeprotection step at the pentasaccharide level, the trisaccharide 313,bearing an acetamido functionality at position 2_(D), was selected asthe donor. Indeed, as it involves the less readily available ECstructure in fewer synthetic steps and does not rely on selectivedeprotection at the 2_(A) position, this path was found particularlyattractive. Again, it relies on the use of appropriately functionalizedknown monosaccharide intermediates (FIG. 13).

The known key di-rhamnoside core structure 322 (Zhang, J.; Mao, J. M.;Chen, H. M.; Cai, M. S. Tetrahedron: Asymmetry 1994, 5, 2283-2290) wasformed by glycosylation of the allyl rhamnoside 314 with thetrichloroacetimidate donor 321 (Castro-Palomino, J. C.; Rensoli, M. H.;Bencomo, V. V. J. Carbohydr. Chem. 1996, 15, 137-146) in the presence ofa catalytic amount of TMSOTf. It should be pointed out that usingdiethyl ether as the solvent, the isolated yield of 322 was 92%, whichcompares favourably with those obtained previously, 60% and 76.2%(Zhang, J.; Mao, J. M.; Chen, H. M.; Cai, M. S. Tetrahedron: Asymmetry1994, 5, 2283-2290), when running the reaction in dichloromethane underpromotion by TMSOTf or BF₃.OEt₂, respectively. De-O-acetylation underZemplén conditions afforded the 2_(A)-O-unprotected acceptor 323 (Pinto,B. M.; Reimer, K. B.; Morissette, D. G.; Bundle, D. R. J. Org. Chem.1989, 54, 2650-2656) in 93% yield.

As shown previously in the construction of the DA intermediate 317, theN-trichloroacetyl trichloroacetimidate 316 appears to be a highlysuitable precursor to residue D when involved in the formation of theβ-GlcNAc linkage at the poorly reactive 2_(A) position. Indeed, reactionof 316 with the acceptor 323 in 1,2-dichloroethane in the presence ofTMSOTf went smoothly and gave the trisaccharide 325 in 96% yield.However, conversion of the N-trichloroacetyl group to the N-acetylderivative 327 was rather less successful as the desired trisaccharidewas obtained in only 42% yield when treated under conditions that hadpreviously been used in the case of a related oligosaccharide (sodiummethoxide, Et₃N, followed by re-N,O-acetylation). (Costachel, C.;Sansonetti, P. J.; Mulard, L. A. J. Carbohydr. Chem. 2000, 19,1131-1150). This result led us to reconsider the protection pattern ofthe glucosamine donor. The N-tetrachlorophthalimide group has beenproposed as an alternative to overcome problems associated with thewidely spread phthalimido procedure when introducing a2-acetamido-2-deoxy-β-D-glucopyranosidic linkage (Debenham, J. S.;Madsen, R.; Roberts, C.; Fraser-Reid, B. J. Am. Chem. Soc. 1995, 117,3302-3303). Thus, the N-tetrachlorophthalimide trichloroacetimidatedonor 324 was selected as an alternative. It was prepared as describedfrom commercially available D-glucosamine (Castro-Palomino, J. C.;Schmidt, R. R. Tetrahedron Lett. 1995, 36, 5343-5346), apart from in thefinal imidate formation step, where we found the use of potassiumcarbonate as base to be more satisfactory than DBU. Glycosylation of 323with 324 in the presence of TMSOTf resulted in the trisaccharide 328 in65% yield. The tetrachlorophthaloyl group was then removed by the actionof ethylenediamine, and subsequent re-N,O-acetylation gave thetrisaccharide 327 in 65% yield. The latter was next converted into thedonor 313 in two steps, analogous to those described for the preparationof 306 from 317. Indeed, de-O-allylation of 327 cleanly gave thehemiacetal 329 (83%), which was then activated into the requiredtrichloroacetimidate (94%). It is worth mentioning that although theyinvolve a different D precursor, both strategies give access to theintermediate 327 in closely related yields, 40 and 42%, respectively.

Initial attempts to form the pentasaccharide 305 from 313 and thepreviously described acceptor 311 (Segat, F.; Mulard, L. A. Tetrahedron:Asymmetry 2002, 13, 2211-2222) in the presence of TMSOTf as promoterwere rather unsuccessful, resulting in at best 17% of the desiredproduct, accompanied by decomposition of the donor into the hemiacetal329 (75%). By using BF₃.OEt₂ as the promoter in place of TMSOTf,reaction of 311 with 313 at room temperature provided 305 in 44% yield,with the acceptor 311 and hemiacetal 329 also recovered in 54% and 29%yield, respectively. We considered that the poor reactivity of theacceptor was responsible for these results, as since the ¹³C NMR of 305,showing several distorted signals (notably C-1_(B), as well as mostcertainly C-3_(C) and C-4_(C)), suggests restricted conformationalflexibility around the position 3_(C). For that matter, the2_(C)-O-acetylated disaccharide 312 was considered as an alternateacceptor. Analogously to the preparation of 311, it was obtained fromthe known diol 330 through regioselective opening of the intermediateorthoester. However, coupling of the potentially less hindered acceptor312 and the trisaccharide donor 313 resulted, at best, in the isolationof the condensation product 331 in 42% yield (not described).

The modest yield of 305 and 331 obtained by this route made thealternative reaction path (FIG. 14) worth investigating, despite themore numerous synthetic steps required. Indeed, it was found ratherappealing when evaluated independently in a closely related series(unpublished results). By this route, a tetrasaccharide acceptor can beformed from two disaccharide building blocks (EC and AB), and coupledwith an appropriate monosaccharide donor as precursor to D. Consideringthat selective deprotection of the 2_(A) hydroxyl group would occur inthe course of the synthesis, glycosylation attempts were limited to the2-O-benzoylated acceptor 311. The disaccharide donor necessary for thispath could be derived from the building block 323, already in hand. Thechoice of temporary protecting group at position 2_(A) was determined byour experience of the stepwise synthesis of the corresponding methylpentasaccharide, (Costachel, C.; Sansonetti, P. J.; Mulard, L. A. J.Carbohydr. Chem. 2000, 19, 1131-1150) where we noted that an acetategroup at this position may not be fully orthogonal to the benzoatelocated at position 2_(C). The chosen group had also to support removalof the anomeric allyl group and the subsequent conversion to thetrichloroacetimidate. At first, a chloroacetate group was anticipated tofulfil these requirements. Thus, the disaccharide 323 was treated withchloroacetic anhydride and pyridine to give the derivative 332 (57%).Anomeric deprotection to give the hemiacetal 333 (84%) and subsequenttrichloroacetimidate activation of the latter into the donor 334 (83%)were performed in the same way as before. Coupling of 311 with 334,carried out in the presence of TMSOTf at −40° C., yielded a complexmixture of products. When the temperature was lowered to −60° C., thecondensation product 338 could be isolated in 22% yield. Alternativedonor protection was attempted. Treatment of 323 with p-methoxybenzylchloride and sodium hydride gave the fully protected derivative 335(97%), which was cleanly converted into the trichloroacetimidate donor337 (82%) in two steps involving the hemiacetal intermediate 336 (73%).Glycosylation of 311 with 337 in the presence of TMSOTf at −40° C. gavethe desired tetrasaccharide 339 in 44% yield. When the temperature waslowered to −60° C., the yield of 339 fell to 34% and a second majorproduct 340 (21%) was observed in the mixture. Indeed, examination ofthe NMR spectra of this product revealed that the pMeOBn group had beenlost. That 340 was the acceptor required for the next step brought theestimated yield of condensation to 55%. Nevertheless, the overalloutcome of this blockwise strategy did not match our expectations, andthis route was abandoned.

Linear strategy to the fully protected pentasaccharide 304 (FIG. 15): Aspreliminary studies have demonstrated, rapid access to suitable buildingblocks allowing the synthesis of higher-order oligosaccharidesrepresentative of fragments of the O—SP of S. flexneri 2a remains achallenge. Major conclusions drawn from our studies favour the design ofa linear synthesis of the target 304. Indeed, when put together with ourprevious work, such as the synthesis of tetrasaccharide 341 (95%)(Costachel, C.; Sansonetti, P. J.; Mulard, L. A. J. Carbohydr. Chem.2000, 19, 1131-1150) or that of trisaccharide 342 (97%) (Segat, F.;Mulard, L. A. Tetrahedron: Asymmetry 2002, 13, 2211-2222), all theabove-described attempted couplings outlined the loss of efficiency ofglycosylation reactions involving rhamnopyranosyl donors glycosylated atposition 2 in comparison to those involving the corresponding acetylateddonor. Thus, matching the linear strategy of the methyl pentasaccharide2 described previously, (Costachel, C.; Sansonetti, P. J.; Mulard, L. A.J. Carbohydr. Chem. 2000, 19, 1131-1150) a synthesis of 304, based ondonors bearing a participating group at O-2, was designed. Three keybuilding blocks were selected. These were the readily accessible ECdisaccharide acceptor 311 benzoylated at C-2 as required for the finalcondensation step leading to the fully protected decasaccharideintermediate; the rhamnopyranosyl trichloroacetimidate 321, which servesas a precursor to residues A and B, and bears a both temporary andparticipating group at position 2; and the trichloroacetamideglucosaminyl donor 316 as a precursor to residue D. As stated above,coupling of 311 and 321 gave 342 in high yield. As observed in themethyl glycoside series, (Costachel, C.; Sansonetti, P. J.; Mulard, L.A. J. Carbohydr. Chem. 2000, 19, 1131-1150) de-O-acetylation using MeONaor methanolic HCl was poorly selective. Although, guanidine/guanidiniumnitrate was proposed as a mild and selective O-deacetylation reagentcompatible with the presence of benzoyl protecting groups, (Ellervik,U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627-1628) none of theconditions tested prevented partial debenzoylation leading to diol 343,as easily confirmed from NMR analysis (not described). The requiredalcohol 310 was readily obtained in an acceptable yield of 84% yield bya five-day acid catalysed methanolysis, using HBF₄ in diethylether/methanol, (Costachel, C.; Sansonetti, P. J.; Mulard, L. A. J.Carbohydr. Chem. 2000, 19, 1131-1150; Pozsgay, V.; Coxon, B. Carbohydr.Res. 1994, 257, 189-215) of the fully protected intermediate 342.Repeating this two-step process using 310 as the acceptor and 321 as thedonor resulted first in the intermediate 344 (90%), and next in thetetrasaccharide acceptor 340 (84%). Glycosylation of the latter with 316gave the fully protected pentasaccharide 304 in high yield (98%), thusconfirming that the combination of the trichloroacetamide participatinggroup and the trichloroacetimidate activation mode in 316 results in apotent donor to be used as a precursor to residue D in the S. flexneriseries, where low-reactive glycosyl acceptors are concerned. Followingthe above described procedure, selective anomeric deprotection of 304furnished the hemiacetal 345 which was smoothly converted to thetrichloroacetimidate donor 346 (66% from 304). From these data, thelinear synthesis of 34, truly benefiting from the use of 321 as a commonprecursor to residue A and B, appears as a reasonable alternative to theblock syntheses which were evaluated in parallel.

Synthesis of the target decasaccharide 301: Having a pentasaccharidedonor in hand, focus was next placed on the synthesis of an appropriatepentasaccharide acceptor. In our recent description of the convergentsynthesis of the B′(E′)C′DAB(E)C octasaccharide, (F. Bélot, C.Costachel, K. Wright, A. Phalipon, L. A. Mulard, Tetrahedron. Lett.2002, 43, 8215-8218) the pentasaccharide 348, bearing a4_(D),6_(D)-O-isopropylidene protecting group, was found a mostconvenient acceptor which encouraged its selection in the present work.Briefly, 348 was prepared in two steps from the known 302. Thus, mildtransesterification of 302 under Zemplén conditions allowed theselective removal of the acetyl groups to give triol 347, which wasconverted to the required acceptor 348 (72% from 302) upon subsequenttreatment with 2-methoxypropene. Relying on previous optimisation of theglycosylation step (Bélot, F.; Costachel, C.; Wright, K.; Phalipon, A.;Mulard, L. A. Tetrahedron. Lett. 2002, 43, 8215-8218), the condensationof 348 and 346 was performed in the presence of a catalytic amount oftriflic acid. However, probably due to the closely related nature of thedonor and acceptor, the reaction resulted in an inseparable mixture ofthe fully protected 349 and the hemiacetal 345 resulting from partialhydrolysis of the donor. Most conveniently, acidic hydrolysis of themixture, allowing the selective removal of the isopropylidene group in349, gave the intermediate diol 350 in a satisfactory yield of 72% forthe two steps. According to the deprotection strategy used for thepreparation of the closely related octasaccharide (Bélot, F.; Costachel,C.; Wright, K.; Phalipon, A.; Mulard, L. A. Tetrahedron. Lett. 2002, 43,8215-8218), diol 350 was engaged in a controlled de-O-acylation processupon treatment with hot methanolic sodium methoxide. However, partialcleavage of the trichloroacetyl moiety, leading to an inseparablemixture, was observed which prevented further use of this strategy.Indeed, it was assumed that besides being isolated and thereforeresistant to Zemplén transacetylation conditions (Liptak, A.; Szurmai,Z.; Nanasi, P.; Neszmelyi, A. Carbohydr. Res. 1982, 99; Szurmai, Z.;Liptak, A.; Snatzke, G. Carbohydr. Res. 1990, 200, 201-208; Szurmai, Z.;Kerékgyarto, J.; Harangi, J.; Liptak, A. Carbohydr. Res. 1987, 174,313-325), the 2_(C)-O-benzoyl groups were most probably highly hinderedwhich contributed to their slow deprotection. Alternatively, 350 wassubmitted to an efficient two-step in-house process involving first,hydrogenolysis under acidic conditions which allowed the removal of thebenzyl groups and second, basic hydrochlorination which resulted in theconversion of the N-trichloroacetyl groups into the required N-acetylones, thus affording 352. Subsequent transesterification gave the finaltarget 301 in 37% yield from 350 (FIG. 16).

D—Synthesis of the 2-Amionoethyl Glycoside of a Hapten Representative ofthe O-specific polysaccharide of Shigella flexneri Serotype 2a and of aCorresponding PADRE-Conjugate

Studies on the recognition of synthetic fragments of the O—SP byprotective homologous monoclonal antibodies suggested that sequenceslarger than one repeating unit were more antigenic, thus probably bettermimicking the natural polysaccharide than shorter ones. Indeed, it isanticipated that better mimics of the O—SP, in terms of bothantigenicity and conformation, would lead to conjugates of higherimmunogenicity. For that reason, the preparation of conjugatescomprising oligosaccharides larger than one repeating unit wasundertaken.

We report herein on the synthesis of the 2-aminoethyl glycosides of ahexasaccharide (402) and on that of the corresponding fully syntheticconjugate (401) using the PADRE as a universal T-helper peptide (seesection E for the background). We have demonstrated that disconnectionat the C-D linkage was appropriate for the construction of largefragments of the S. flexneri 2a O—SP (F. Bélot, K. Wright, C. Costachel,A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074). Based onour experience in the field, a strategy to target 401, implicating theDAB(E)C building block bearing the required acetamido function atposition 2_(D) (406) as donor and the recently disclosed acceptor 405(K. Wright, C. Guerreiro, I. Laurent, F. Baleux, L. A. Mulard, Org.Biomol. Chem. 2004, 2, 1518-1527) as a precursor to the spacer-armed Dresidue (FIG. 17). Although permanent blocking of OH-4_(D) and OH-6_(D)with an isopropylidene acetal may appear somewhat unusual, this choicewas a key feature of the strategy. It was based on former observationsin the methyl glycoside series, demonstrating that its use couldovercome some of the known drawbacks of the corresponding benzylideneacetal, (Bundle, D. R.; Josephson, S. Can. J. Chem. 1979, 57, 662-668;Mulard, L. A.; Costachel, C.; Sansonetti, P. J. J. Carbohydr. Chem.2000, 19, 849-877) including its poor solubility.

Synthesis of the hexasaccharide 402 (FIG. 18): The key pentasaccharidedonor 406 was obtained from the recently disclosed precursor 407 (seesection F, compound 611). The latter was converted to the hemiacetal 408following a two-step process including Iridium complex promotedisomerisation of the allyl moiety into the propen-1-yl, (Oltvoort, J.J.; van Boeckel, C. A. A.; der Koning, J. H.; van Boom, J. Synthesis1981, 305-308) and hydrolysis of the latter upon treatment with aqueousiodine (Nashed, M. A.; Anderson, L. J. Chem. Soc. Chem. Commun. 1982,1274-1282). Subsequent reaction of 408 with trichloroacetonitrile in thepresence of catalytic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) cleanlygave the trichloroacetimidate donor 406 (85% from 407). Previousglycosidation attempts in the series indicated that when run at lowtemperature or room temperature, reactions using the D acceptor 405occasionally resulted in a rather poor yield of the condensationproduct. This was tentatively explained by the still rather poorsolubility of 405. When using 1,2-dichloroethane (1,2-DCE) as thesolvent, the condensation could be performed at higher temperature,which proved rewarding. Indeed, optimized coupling conditions relied onthe concomitant use of a catalytic amount of triflic acid in thepresence of 4 Å molecular sieves as the promoter and 1,2-DCE as thesolvent, while the condensation was performed at 80° C. The fullyprotected hexasaccharide 409 was isolated in a satisfactory 78% yield.That the hemiacetal 408, resulting from the hydrolysis of the excessdonor could be recovered was of great advantage is one considers scalingup the process (not described). Acidic hydrolysis of the isopropylideneacetal smoothly converted 409 into the corresponding diol 410 (94%).Resistance of isolated benzoyl groups to Zemplén transesterification hasbeen reported (Lipták, A.; Szurmai, Z.; Nanasi, P.; Neszmelyi, A.Carbohydr. Res. 1982, 99, 13-21, Szurmai, Z.; Kerékgyarto, J.; Harangi,J.; Lipták, A. Carbohydr. Res. 1987, 174, 313-325 Szurmai, Z.; Lipták,A.; Snatzke, G. Carbohydr. Res. 1990, 200, 201-208). It was alsoobserved previously in the series, upon attempted removal of a benzoylgroup located at position 2_(C) (F. Bélot, K. Wright, C. Costachel, A.Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074). Again, the2_(C)-O-benzoyl group in 410 was particularly resistant to Zemplénde-O-acylation, and successful transesterification required a week. Inthat case, heating was avoided in order to prevent any potentialmigration of the acyl group which would lead to the N-deacylatedproduct. Conversion of the hexaol 411 into the target 402 (77%) wassuccessfully accomplished upon concomitant hydrogenolysis of theremaining benzyl protecting group and reduction of the azido moiety intothe corresponding amine. As observed earlier, the latter was bestperformed under acidic conditions.

Synthesis of the fully synthetic glycoconjugate 401 (FIG. 17):4-(N-maleimido)-n-butanoyl was selected as the linker, and incorporatedusing commercially available 404 by covalent linkage to the side chainamino group of a Lysine residue added at the C-terminus of the PADREsequence (PADRE-Lys). The latter was assembled using standard Fmocchemistry for solid-phase peptide synthesis (Chan, W. C.; White, P. D.Fmoc solid phase peptide synthesis; Oxford University Press: New York,2000). Standard side chain protecting groups were used, except for thatof the C-terminal Lysine side chain which was protected by the1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) group(Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.;Chan, W. C. Tetrahedron Lett. 1998, 39, 1603-1606) to allow specificintroduction of the maleïmide group. The thiol functionality wasintroduced onto the carbohydrate haptens as a masked thiol function(acetylthioester), which is easily generated in situ during theconjugation process Thus, reaction of 402 with S-acetylthioglycolic acidpentafluorophenyl ester (SAMA-oPfp) resulted in the site-selectiveelongation of the aminoethyl spacer via a thioacetyl acetamido linker.Derivatization could be monitored by RP-HPLC with detection at 215 nm.Under these conditions, the required thioacetyl-armed intermediate, 412was isolated in 53% yield. Its structure was confirmed based on MS andNMR analysis. Conjugation of the carbohydrate haptens to the maleimidoactivated PADRE-Lys (403) was run in phosphate buffer at pH 6.0 inpresence of hydroxylamine (H. F. Brugghe, H. A. M. Timmermans, L. M. A.van Unen, G. J. T. Hove, G. W. der Werken, J. T. Poolman, P. Hoogerhout,Int. J. Peptide Protein Res. 1994, 43, 166) and monitored by RP-HPLC.Lastly, RP-HPLC purification gave the target neoglycopeptide 401 as asingle product, whose identity was assessed based on MS analysis, inyields of 58%.

E—Preparation of Chemically Defined Glycopeptides as Potential SyntheticConjugate Vaccines Against Shigella flexneri Serotype 2a Disease

The target neoglycopeptides were constructed by covalently linking ashort peptide, serving as a T-helper epitope, to appropriatecarbohydrate haptens, serving as B epitopes mimicking the S. flexneri 2aO—Ag. Our approach is based on rational bases involving a preliminarystudy of the interaction between the bacterial O—SP and homologousprotective monoclonal antibodies, which helped to define thecarbohydrate haptens.

Fragments ECD, B(E)CD and AB(E)CD were selected as haptens that will actas B-epitopes in the conjugates. Three fully synthetic linearneoglycopeptides 501, 502 and 503, corresponding to haptens ECD, B(E)CD,and AB(E)CD, respectively, were synthesized according to a strategybuilt up on the concept of chemoselective ligation which allows theselective one-point attachment of the free B and T epitopes in aqueousmedia. All conjugates involve the peptide PADRE (J. Alexander, J.Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal, K. Snoke, H. M.Serra, R. T. Kubo, A. Sette, H. M. Grey, Immunity 1994, 1, 751-761; J.Alexander, A.-F. d. Guercio, A. Maewal, L. Qiao, J. Fikes, R. W.Chesnut, J. Paulson, D. R. Bundle, S. DeFrees, A. Sette, J. Immunol.2000, 164, 1625-1633) as the universal T-cell epitope.

Retrosynthetic analysis of the saccharidic haptens (FIG. 19): Analysisof S. flexneri 2a O—SP suggests that, due to the 1,2-cis glycosidiclinkage involved, construction of the EC disaccharide is probably themost demanding. Besides, prior work in this laboratory has shown thatthe C-D glycosidic linkage was an appropriate disconnection site whendealing with the blockwise synthesis of oligosaccharide fragments of S.flexneri O-2a SP. (F. Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002,13, 2211-2222) These observations supported the design of a syntheticstrategy common to all three targets. Basically, it relies on (i) thecondensation of an EC (504), (C. Costachel, P. J. Sansonetti, L. A.Mulard, J. Carbohydr. Chem. 2000, 19, 1131-1150) B(E)C (505) (F. Bélot,C. Costachel, K. Wright, A. Phalipon, L. A. Mulard, Tetrahedron. Lett.2002, 43, 8215-8218) or AB(E)C (506) donor to a D acceptor (507),functionalized at the anomeric position with an azidoethyl spacer; (ii)elongation of the spacer with introduction of a masked thiol group toallow its coupling onto a PADRE peptide derivatized by a maleimido groupon a C-terminal Lysine (508). The carbohydrate synthesis relies on thetrichloroacetimidate methodology and the use of known building blockswhenever possible.

Synthesis of the aminoethyl ECD building block 518 (FIG. 20): The noweasily accessible disaccharide donor 504, with a benzoyl participatinggroup at position 2_(C), was used as the precursor to the EC moiety inthe construction of 501. It was prepared, as described, (Costachel, C.;Sansonetti, P. J.; Mulard, L. A. J. Carbohydr. Chem. 2000, 19,1131-1150) in 5 steps and 45% overall yield from2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl trichloroacetimidate (509) (R.R. Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem. 1984 1343-1357; R. R.Schmidt, J. Michel, Tetrahedron Lett. 1984, 25, 821-824) and allyl2,3-O-isopropylidene-α-L-rhamnopyranoside (510) (R. Gigg, S. Payne, R.Conant, J. Carbohydr. Chem. 1983, 2, 207-223) through the keyintermediate diol 511 (69% from 510). Introduction of the azidoethylspacer on a glucosaminyl intermediate was performed according to a knownprocedure (Eklind, T.; Gustafsson, R.; Tidén, A.-K.; Norberg, T.; Åberg,P.-M. J. Carbohydr. Chem. 1996, 15, 1161-1174) by coupling ofazidoethanol onto the oxazoline 512 to give the triacetate 513. (T.Eklind, R. Gustafsson, A.-K. Tidén, T. Norberg, P.-M. Åberg, J.Carbohydr. Chem. 1996, 15, 1161-1174) We have shown on several occasionsin the S. flexneri series, that regioselective protection of the 4- and6-OH groups of precursors to residue D with an isopropylidene acetal wasappropriate, especially when such precursors are involved in a blockwisesynthesis based on the disconnection at the C-D linkage. Thus, Zempléndeacetylation of 513 gave the triol 514 which was converted to the keyacceptor 507 (81% from 513) upon reaction with 2,2-dimethoxypropaneunder acid catalysis. When the latter was glycosylated with the donor504 in the presence of BF₃.OEt₂ in CH₂Cl₂, the fully protectedtrisaccharide 515 was isolated in 58% yield together with the diol 516(30%), resulting from partial loss of the isopropylidene acetal. When504 and 507 were glycosylated in the presence of a catalytic amount ofTMSOTf, no side-reaction was observed, and the condensation product 515was obtained in 86% yield. Quantitative conversion of 515 into 516 wasmore conveniently achieved by acidic hydrolysis of the former with 95%aq TFA. Debenzoylation of 516 gave the tetraol 517 (94%) which wassubsequently transformed into the aminoethyl-trisaccharide 518 (69%) byhydrogenation in the presence of palladium-on-charcoal (Pd/C) and 1M aqHCl to convert the formed amine to its hydrochloride salt. Indeed,others have pointed out that hydrogenolysis using Pd/C in the presenceof a free amine was sluggish and low-yielding (Stahl, W.; Sprengard, U.;Kretschmar, G.; Kunz, H. Angew. Chem. Int. Ed. 1994, 33, 2096-2098;Spikjer, N. M.; Keuning, C. A.; Hooglugt, M. Tetrahedron 1996, 52,5945-5960; Li, Q.; Li, H.; Lou, Q.-H.; Su, B.; Cai, M.-S.; Li, Z.-J.Carbohydr. Res. 2002, 337, 1929-1934). In order to prevent anyside-reaction at a latter stage of the synthesis, isolation of pure 518was subsequently submitted to reversed-phase HPLC (RP-HPLC).

Synthesis of the aminoethyl B(E)CD building block 525 (FIG. 21): Theknown rhamnopyranosyl tricholoracetimidate 520, acetylated at its 2-,3-, and 4-OH groups thus acting as a chain terminator, was chosen as theprecursor to residue B. Benzoylation of diol 511 to give 519 wasperformed by regioselective opening of the cyclic orthoesterintermediate as described (Segat, F.; Mulard, L. A. Tetrahedron:Asymmetry 2002, 13, 2211-2222). Glycosylation of the latter by donor520, with activation by a catalytic amount of TMSOTf proceeded smoothlyin Et₂O to yield the fully protected trisaccharide 521 (89%), which wasde-O-allylated into the hemiacetal 522 (80%) following a two stepprocess involving (i) iridium(I)-catalysed isomerisation of the allylglycoside to the prop-1-enyl glycoside (Oltvoort, J. J.; van Boeckel, C.A. A.; der Koning, J. H.; van Boom, J. Synthesis 1981, 305-308) and (ii)subsequent hydrolysis (Gigg, R.; Payne, S.; Conant, R. J. Carbohydr.Chem. 1983, 2, 207-223; Gigg, R.; Warren, C. D. J. Chem. Soc. C 1968,1903-1911). The selected trichloroacetimidate leaving group wasintroduced by treatment of 522 with trichloroacetonitrile in thepresence of a catalytic amount of DBU, which resulted in the formationof 505 (99%). Condensation of the latter with acceptor 507 was performedin CH₂Cl₂ in the presence of a catalytic amount oftrifluoromethanesulfonic acid (TfOH) to give the requiredtetrasaccharide 523 (76%). Acidic hydrolysis of the latter using 95% aqTFA gave the intermediate diol 524 in 95% yield. Deacylation of theresulting diol under Zemplén conditions followed by debenzylation andconcomitant conversion of the azide into the corresponding amine to givethe key aminoethyl-armed tetrasaccharide 525 (77%) was performed bytreatment of 524 with hydrogen in the presence of Pd/C under acidicconditions. Again, compound 525 was purified by RP-HLPC beforeelongation of the spacer or conjugation.

Synthesis of the aminoethyl AB(E)CD building block 537 (FIG. 22): Thesynthesis of 537 is based on the condensation of acceptor 507 and donor506, which resulted from the selective deallylation and anomericactivation of the key intermediate tetrasaccharide 533. The latter wasobtained according to two routes following either a block strategy(route 1) based on the condensation of an AB disaccharide donor (530)and the EC disaccharide acceptor 519, or a linear strategy (route 2)involving the stepwise elongation of 519. The construction of the donor530 was based on the use of the known allyl rhamnopyranoside 526(Westerduin, P.; der Haan, P. E.; Dees, M. J.; van Boom, J. H.Carbohydr. Res. 1988, 180, 195-205), having permanent protecting groupsat position 3 and 4, as the precursor to residue B, and thetrichloroacetimidate chain terminator 527 (Ziegler, T.; Bien, F.;Jurish, C. Tetrahedron: Asymmetry 1998, 9, 765-780), acting as aprecursor to residue A. Condensation of the two entities in the presenceof a catalytic amount of TMSOTf resulted in the fully protected 528(96%), which was selectively de-O-allylated into 529 (84%) according tothe protocol described above for the preparation of 522. Subsequenttreatment of 529 with trichloroacetonitrile and a catalytic amount ofDBU gave the required 530 (96%). Glycosylation of 519 with the latterunder TMSOTf promotion afforded the fully protected tetrasaccharide 533in 55% yield. No β-anomer was detected. Route 1 was considered initiallyin order to prevent extensive consumption of the EC disaccharide 511.Given the relatively low yield of coupling of 519 and 530, route 2 wasconsidered as well. Of all precursors to 534, only that to residue B,namely the donor and potential acceptor 531, differed from those used inroute 1. Conventional glycosylation of disaccharide 519 and 531 andsubsequent selective deacetylation using methanolic HBF₄, gave theacceptor 532 in 70% yield from 519. The trisaccharide 532 wasglycosylated with trichloroacetimidate 527 in an analogous fashion toglycosylation of 519 with 530, yielding 533 (92%). Anomericde-O-allylation of this key intermediate, as described above for thepreparation of 522, gave the corresponding hemiacetal 534 (90%) whichwas converted into the required trichloroacetimidate 506 (88%) upontreatment with trichloroacetonitrile and DBU. Condensation of donor 506with the glucosaminyl acceptor 507 was performed under promotion by TfOHor TMSOTf; which resulted in the fully protected pentasaccharide 535 in62% and 80% yield, respectively. Following the process described for thepreparation of 525, compound 535 was submitted to acetolysis (97%) andsubsequent Zemplén deacylation to give the partially deblocked 536(87%), which was next converted to the aminoethyl-spacer pentasaccharide537 upon treatment with hydrogen in the presence of Pd/C. Final RP-HPLCpurification resulted in the isolation of 537 in 53% yield.

Synthesis of the target neoglycopeptides 501-503 (FIG. 23): In allcases, chemoselective ligation of the B and T epitopes was achievedthrough coupling of the carbohydrate haptens pre-functionalized with athiol function and a maleimido group properly introduced at the Cterminus of the T helper peptide. Such a strategy was chosen in order toexploit the high reactivity and specificity of thiol groups towards themaleimide functionality (Marrian, D. H. J. Chem. Soc. C 1949, 1515),which allows specific and high-yielding modification of the former inthe presence of other nucleophiles (Hermanson, G. T. Bioconjugatetechniques; Academic Press: New York, 1996). It was used previouslyunder various forms in the coupling of carbohydrate haptens to eitherproteins (Ragupathi, G.; Koganty, R. R.; Qiu, D.; Llyod, K. O.;Livingston, P. O. Glycoconjugate J. 1998, 15, 217-221; Shin, I.; Jung,H.; Lee, M. Tetrahedron Lett. 2001, 42, 1325-1328) or peptides (Kandil,A.; Chan, N.; Klein, M.; Chong, P. Glycoconjugate J. 1997, 14, 13-17).To our knowledge, in all the reported cases the maleimide functionalitywas introduced onto the carbohydrate hapten. On the contrary, ourstrategy relies on the introduction of this activating group on the Thelper peptide. The immunogenicity of various maleimide-derived couplingreagents was evaluated in a model system. Based on the reported data,(Peeters, J. M.; Hazendonk, T. G.; Beuvery, E. C.; Tesser, G. I. J.Immunol. Methods 1989, 120, 133-143) 4-(N-maleimido)-n-butanoyl wasselected as the linker, and incorporated by covalent linkage to the sidechain amino group of a Lysine residue added at the C-terminus of thePADRE sequence (PADRE-Lys). It is worth mentioning that the strategydescribed herein somewhat differs from that described by others whendemonstrating the usefulness of PADRE in the construction of immunogenicneoglycopeptides (Alexander, J.; Guercio, A.-F. d.; Maewal, A.; Qiao,L.; Fikes, J.; Chesnut, R. W.; Paulson, J.; Bundle, D. R.; DeFrees, S.;Sette, A. J. Immunol. 2000, 164, 1625-1633).

The Lysine-modified PADRE was assembled using standard Fmoc chemistryfor solid-phase peptide synthesis (Chan, W. C.; White, P. D. Fmoc solidphase peptide synthesis; Oxford University Press: New York, 2000).Standard side chair protecting groups were used, except for that of theC-terminal Lysine side chain which was protected by the1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) group(Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.;Chan, W. C Tetrahedron Lett. 1998, 39, 1603-1606). Indeed, thisorthogonal protecting group strategy allows specific introduction of themaleïmide group on the C-terminal Lysine, upon selective cleavage of theivDde by hydrazine. The thiol functionality was introduced onto thecarbohydrate haptens as a masked thiol function (acetylthioester), whichis easily generated in situ during the conjugation process. Thus,reaction of 518, 525, and 537 with S-acetylthioglycolic acidpentafluorophenyl ester (SAMA-θPfp) resulted in the site-selectiveelongation of their aminoethyl spacer via a thioacetyl acetamido linkerDerivatization could be monitored by RP-HPLC with detection at 215 nm.Under these conditions, the required thioacetyl-armed intermediates,538, 539 and 540 were isolated in 53%, 74%, and 75% yield, respectively.Their structure was confirmed based on MS and NMR analysis. Conjugationof the carbohydrate haptens to the maleimido activate PADRE-Lys (508)was run in phosphate buffer at pH 6.0 in presence of hydroxylamine H. F.Brugghe, H. A. M. Timmermans, L. M. A. van Unen, G. J. T. Hove, G. W.der Werken J. T. Poolman, P. Hoogerhout, Int. J. Peptide Protein Res.1994, 43, 166-172) and monitored by RP-HPLC. Lastly, RP-HPLCpurification gave the target neoglycopeptides 501, 502, and 503 assingle products, which identity was assessed based on MS analysis, inyields of 58%, 48% and 46%, respectively.

F—Synthesis of Two Linear PADRE-Conjugates Bearing a Deca- orPentadecasaccharide B epitope as potential synthetic vaccine againstShigella flexneri Serotype 2a Infection

We report herein on the synthesis of the PADRE conjugates of adeca-(601) and a pentadecasaccharide (602), corresponding to a dimer[AB(E)CD]₂ and a trimer [AB(E)CD]₃ of the branched pentasaccharide I,respectively (FIG. 24). The synthesis is based on a modular approachinvolving three partners. Basically, it relies on (i) the use ofappropriate haptens functionalized at the anomeric position with anaminoethyl spacer, 603 and 604, respectively; (ii) the incorporation ofa thioacetyl acetamido linker as a masked thiol functionality, and (iii)the use of a PADRE peptide derivatized by a maleimido group on aC-terminal lysine (605).

Considering the targets 603 and 604, a disconnection at the D-A linkagewould appear most appropriate. However, others have shown that such adisconnection strategy was not suitable even when involving di- ortrisaccharide building blocks (B. M. Pinto, K. B. Reimer, D. G.Morissette, D. R. Bundle, J. Org. Chem. 1989, 54, 2650; B. M. Pinto, K.B. Reimer, D. G. Morissette, D. R. Bundle, Carbohydr. Res. 1990, 196,156), thus this route was avoided. More recently, disconnections at theA-B, B-C and C-D linkages were evaluated in this laboratory whensynthesizing successfully the methy glycoside of the frame-shifteddecasaccharide D′A′B′(E′)C′DAB(E)C (F. Bélot, K. Wright, C. Costachel,A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074). It wasdemonstrated on that occasion that disconnection at the C-D linkage wasindeed appropriate for the construction of large fragments of the S.flexneri 2a O—SP. Based on our experience in the field, we designed ablockwise strategy to targets 603 and 604, implicating an AB(E)Ctetrasaccharide donor (606), a DAB(E)C potential acceptor acting as adonor (607), and the recently disclosed acceptor 608 (K. Wright, C.Guerreiro, I. Laurent, F. Baleux, L. A. Mulard, Org. Biomol. Chem. 2004,2, 1518-1527), bearing a masked aminoethyl spacer, as a precursor to thereducing end D residue (FIG. 24). Although permanent blocking ofOH-4_(D) and OH-6_(D) with an isopropylidene acetal may appear somewhatunusual, this choice was a key feature of the strategy. It was based onformer observations in the methyl glycoside series, demonstrating thatits use could overcome some of the known drawbacks of the correspondingbenzylidene acetal (F. Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002,13, 2211-2222) J. Banoub, D. R. Bundle, Can. J. Chem. 1979, 57, 2091),including its poor solubility. In order to reduce the number ofsynthetic steps, it was found appropriate to access the AB(E)C donor andthe DAB(E)C building block from a common key AB(E)C tetrasaccharideintermediate 609. Most of all, the design of the pentasaccharidebuilding block 607 was a key element to success. Indeed, a leadingconcept of the overall strategy was to limit the number oftransformations at later stages in the syntheses. Concerning the choiceof 607, the reader's attention is thus drawn to (i) the permanentblocking of position 4_(D) and 6_(D) as an isopropylidene acetal, (ii)the introduction of a participating benzoyl group, resistant to Zempléndeacylation, at position 2_(A), (iii) the temporary protection ofposition 3_(D) as an orthogonal acetate, (iv) the early introduction ofthe required 2_(D) acetamido functionality, and (v) the activation ofthe anomeric position as a trichloroacetimidate. Indeed, it should beoutlined that the syntheses disclosed herein are based on the use of thetrichloroacetimidate (TCA) chemistry, (R. R. Schmidt, W. Kinzy, Adv.Carbohydr. Chem. Biochem. 1994, 50, 21-123) and that known buildingblocks were used whenever possible.

Synthesis of the tetrasaccharide building block 606 (FIG. 25):Preparation of 606 was conveniently achieved from the previouslydescribed tetrasaccharide 609, (F. Bélot, K. Wright, C. Costachel, A.Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074) in a nonoptimized yield of 56%, according to a conventional protocol, namelyselective removal of the anomeric allyl group and subsequent activationupon reaction of the resulting hemiacetal with trichloroacetonitrile inthe presence of catalytic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

Synthesis of the pentasaccharide building block 607 (FIG. 25): Startingfrom 609, we recently described the synthesis of the DAB(E)C buildingblock 610 bearing a trichloroacetamide function at position 2_(D). Thiscrucial intermediate could be obtained in high yield when running thecondensation on a 5 g scale. It was used successfully as the donor inthe synthesis of the D′A′B′(E′)C′DAB(E)C decasaccharide, once convertedto the corresponding trichloroacetimidate. (F. Bélot, K. Wright, C.Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074)However, for the present purpose we reasoned that conversion of thetrichloroacetamide moiety into the required acetamide at an early stagein the synthesis was preferable. Thus, reductive free-radicaldechlorination of 610 using Bu₃SnH in the presence of catalytic AIBNallowed the conversion of the N-trichloroacetyl moiety into N-acetyl, togive the known 611 (68%), previously obtained according to analternative and somewhat lower yielding strategy (F. Bélot, K. Wright,C. Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69,1060-1074). Controlled de-O-acetylation of 611 under Zemplén conditionsgave the triol 612, which was next converted to the correspondingalcohol 613 upon reaction with 2,2-dimethoxypropane (81% from 611).Conventional acetylation at position 3_(D) then gave the fully protectedintermediate 614 (94%), the good overall yield of this three-stepconversion (611→614, 76%) outlining its interest. The latter wastransformed into the hemiacetal 615 (82%) following a two-step processincluding Iridium complex promoted isomerisation of the allyl moietyinto the corresponding propen-1-yl (J. J. Oltvoort, C. A. A. vanBoeckel, J. H. der Koning, J. van Boom, Synthesis 1981, 305), andhydrolysis of the latter upon treatment with mercuric chloride, since itwas originally demonstrated that labile isopropylidene groups werestable to such neutral conditions (R. Gigg, C. D. Warren, J. Chem. Soc.C 1968, 1903). Subsequent reaction of 615 with trichloroacetonitrile inthe presence of catalytic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)cleanly gave the key building block 607 (85% from 614).

Synthesis of the aminoethyl decasaccharide 603 (FIG. 26): Previousglycosidation attempts in the series indicated that when run at lowtemperature or room temperature, reactions using the D acceptor 608occasionally resulted in a somewhat poor yield of the condensationproduct. This was tentatively explained by the still rather lowsolubility of 608. When using 1,2-dichloroethane (1,2-DCE) as thesolvent, the condensation could be performed at higher temperature,which proved rewarding. Indeed, optimized coupling conditions of 607 and608, used in slight excess, relied on the concomitant use of a catalyticamount of triflic acid in the presence of 4 Å molecular sieves as thepromoter and 1,2-DCE as the solvent, while the condensation wasperformed at 75° C., according to a known protocol (F. Bélot, D. Rabuka,M. Fukuda, O. Hindsgaul, Tetrahadron Lett. 2002, 43, 7743) which hadrecently been adapted to the use of acceptor 608 in the S. flexneriseries. The fully protected hexasaccharide 616 was isolated in asatisfactory 76% yield. The resistance of the two isopropylidene acetalsto the harsh acidic conditions of the glycosidation reaction isnoteworthy. That the hemiacetal 615, resulting from the hydrolysis ofthe excess donor could be recovered was of great advantage if oneconsiders scaling up the process (not described). Resistance of isolatedbenzoyl groups to Zemplén transesterification has been reported (A.Liptak, Z. Szurmai, P. Nanasi, A. Neszmelyi, Carbohydr. Res. 1982, 99;Z. Szurmai, J. Kerékgyarto, J. Harangi, A. Liptak. Carbohydr. Res. 1987,174, 313; Z. Szurmai, A. Liptak, G. Snatzke, Carbohydr. Res. 1990, 200,201). It was also observed previously in the series, upon attemptedremoval of a benzoyl group located at position 2_(C). Thus, asanticipated selective deacetylation at the 3-OH of the non reducingresidue, gave the D′AB(E)CD acceptor 617 in a yield of 97%, whichconfirmed the orthogonality of the various protecting groups in use atthis stage. Condensation of the latter and 606 was performed in 1,2-DCEusing triflic acid as the promoter. One may note that although thecondensation involves the construction of the C-D linkage, thus somewhatresembling the preparation of the hexasaccharide 616, heating was notrequired and the glycosylation went smoothly at 10° C. to give the fullyprotected decasaccharide 618 (82%). Acidic hydrolysis of the acetalsgave the tetraol 619 (75%). Transesterification of the acyl groups wasbest performed by overnight heating of 619 in methanolic sodiummethoxide. Final hydrogenolysis of the benzyl groups and concomitantconversion of the azido group into the corresponding amine gave thetarget 603 (71% from 619). As observed earlier, (Q. Li, H. Li, Q.-H.Lou, B. Su, M.-S. Cai, Z.-J. Li, Carbohydr. Res. 2002, 337, 1929) thelatter transformation was best performed under acidic conditions.

Synthesis of the aminoethyl pentadecasaccharide 604 (FIG. 27): Therather convenient access to the building block 607 allowed the targetingof larger sequences. Thus, having the hexasaccharide acceptor 617 inhands, the two-step glycosylation/deacetylation process involving 607was repeated. Analogously to the condensation step leading to the fullyprotected decasaccharide, condensation of 617 and the pentasaccharidedonor 607 in the presence of triflic acid was run at a temperature below5° C. Under such conditions, the fully protected undecasaccharide 621was isolated in an excellent yield of 90%, outlining once more thecompatibility of rather labile isopropylidene groups with theglycosylation conditions in use. Zemplén transesterification at the nonreducing 3_(D)-OH of the latter, resulting in the required acceptor 622(91%), proved as efficient. Condensation of this key intermediate withthe tetrasaccharide trichloroacetimidate donor 606 was performedaccording to the same protocol, using triflic acid as the promoter. Thefully protected pentadecasaccharide 623 was isolated in a satisfactoryyield of 82%. Conversion of 623 to the target 604 was performed byrunning the stepwise sequence described for the preparation of 603.Acidic hydrolysis of the isopropylidene groups afforded the hexaol 624(83%). Again, running the transesterification step at high temperatureallowed to overcome the resistance of the isolated 2_(C)-benzoyl groupsto methanolic transesterification. Lastly, conventional hydrogenolysisof the benzyl groups and concomitant reduction of the azide moietyallowed the smooth conversion of de-O-acylated intermediate into thepentadecasaccharide hapten 604 (65% from 624). Interestingly, althoughthe number of synthetic steps involved may be somewhat challenging,those are in average high yielding, making large amounts of 604reachable.

Synthesis of the target conjugates 601 and 602 (FIG. 24): Chemoselectiveligation of the carbohydrate B and peptide T epitopes was achievedthrough coupling of the carbohydrate haptens pre-functionalized with athiol function and a maleimido group properly introduced at the Cterminus of the T helper peptide, which allows specific andhigh-yielding modification of the former in the presence of othernucleophiles (G. T. Hermanson, Bioconjugate techniques, Academic Press,New York, 1996). Based on reported data on the immunogenicity of variousmaleimide-derived coupling agents (J. M. Peeters, T. G. Hazendonk, E. C.Beuvery, G. I. Tesser, J. Immunol. Methods 1989, 120, 133),4-(N-maleimido)-n-butanoyl was selected as the linker. It was covalentlylinked to the side chain amino group of a lysine residue added to theC-terminus of the PADRE sequence (PADRE-Lys) according to an in-houseprocess (K. Wright, C. Guerreiro, I. Laurent, F. Baleux, L. A. Mulard,Org. Biomol. Chem. 2004, 2, 1518), differing from that describedpreviously by others (J. Alexander, A.-F. d. Guercio, A. Maewal, L.Qiao, J. Fikes, R. W. Chesnut, J. Paulson, D. R. Bundle, S. DeFrees, A.Sette, J. Immunol. 2000, 164, 1625). Reaction of 603 and 604 withS-acetylthioglycolic acid pentafluorophenyl ester (SAMA-Pfp) resulted inthe site-selective elongation of their aminoethyl spacer with athioacetyl acetamido linker, yielding 620 (FIG. 26) and 625 (FIG. 27) in61% and 63% yield, respectively. Derivatization could be monitored byRP-HPLC with detection at 215 nm and structure confirmation was based onMS and NMR analysis. Conjugation of the carbohydrate haptens to themaleimido activated PADRE-Lys (605) was run in phosphate buffer at pH6.0 in the presence of hydroxylamine (H. F. Brugghe, H. A. M.Timmermans, L. M. A. van Unen, G. J. T. Hove, G. W. der Werken, J. T.Poolman, P. Hoogerhout, Int. J. Peptide Protein Res. 1994, 43, 166) andmonitored by RP-HPLC. Lastly, RP-HPLC purification gave the targetneoglycopeptides 601 and 602 as single products, whose identity wasassessed by MS analysis, in yields of 44% and 67%, respectively.

G. Synthesis of Biotinylated Analogues of OligosaccharidesRepresentative of Fragments of the O—SP of S. flexneri 2a

The tri- (ECD), tetra- (B(E)CD), penta- (AB(E)CD), hexa-(D′AB(E)CD),deca- ({AB(E)CD}₂) and pentadecasaccharide ({AB(E)CD}₃) were synthesizedas their biotine conjugates 708-713, respectively (FIG. 28). Analogouslyto that used for the preparation of the corresponding glycopeptides, thesynthetic strategy relied on a chemoselective ligation step between acommercially available maleimide-activated biotine derivative 707 andthe saccharides functionalized as thiols. The known thioacetates701-703, disclosed in our reports on the synthesis of thePADRE-conjugates (K. Wright, C. Guerreiro, I. Laurent, F. Baleux, L. A.Mulard, Org. Biomol. Chem. 2004, 2, 1518), 704 (see part D, compound413), and 705-706 (see part F, compounds 620 and 625, respectively) wereused as precursors to the required thiols. Accordingly, conjugation ofthe carbohydrate haptens to the maleimido activated biotine (707) wasrun in phosphate buffer at pH 6.0 in presence of hydroxylamine (H. F.Brugghe, H. A. M. Timmermans, L. M. A. van Unen, G. J. T. Hove, G. W.der Werken, J. T. Poolman, P. Hoogerhout, Int. J. Peptide Protein Res.1994, 43, 166) and monitored by RP-HPLC. Lastly, RP-HPLC purificationgave the target conjugates as single products, whose identity wasassessed based on MS analysis.

H. Synthesis of a Shigella flexneri 2a Pentasaccharide-PADRE ConjugateUsing an Alternate Conjugation Chemistry

We report herein on the synthesis of the(2-bromoethyl)carbonylaminoethyl glycoside of the pentasaccharideAB(E)CD (802) and on that of the corresponding fully synthetic conjugate(801) using the PADRE as a universal T-helper peptide (see section E forthe background). The target 801 was obtained by chemoselective ligationof 802 to the side chain thiol group of a cysteine residue added at theC-terminus of the PADRE sequence (PADRE-Cys, 803).

(3-Bromopropionyl) was selected as the linker, and incorporated usingthe succinimidyl intermediate 804, itself prepared in one step fromcommercially available 3-bromopropionic acid (86%). Thus, reaction of805 with 804 resulted in the site-selective elongation of the aminoethylspacer via a 3-bromopropionyl linker. Derivatization could be monitoredby RP-HPLC with detection at 215 nm. Under these conditions, theintermediate 802 was isolated in 69% yield. Its structure was confirmedbased on MS and NMR analysis (not described). The PADRE-Cys sequence wasassembled using standard Fmoc chemistry for solid-phase peptidesynthesis (Chan, W. C.; White, P. D. Fmoc solid phase peptide synthesis;Oxford University Press: New York, 2000). Standard side chain protectinggroups were used. Conjugation of the carbohydrate hapten 802 to thePADRE-Cys (803) was run in anhydrous DMF and monitored by RP-HPLC.Lastly, preparative RP-HPLC purification gave the target neoglycopeptide801 (57%) as a single product, whose identity was assessed based on MSanalysis.

EXPERIMENTAL Legend of Figures

FIG. 1: Synthesis of the linear ECDAB-OMe pentasaccharide 101 from thecompounds 104, 109, 113 and 114 according to steps a) to j).

FIG. 2: Retrosynthetic analysis of pentasaccharide 102 implying thesynthons 118, 119 and 113.

FIG. 3: Synthesis of the trisaccharide 125 (intermediate for thesynthesis of the pentasaccharide 102

FIG. 4: Synthesis of the AB(E)CD pentasaccharide 102 from compound 127,via compounds 128, 120, 105, 125, 129, 130, 131, 132, 133, and 134according to steps a) to j).

FIG. 5: Representation of the orthoester 135

FIG. 6: Synthesis of the B(E)CD tetrasaccharide 103

FIG. 7: Pentasaccharides 201 (DAB(E)C), 202, 203

FIG. 8: Synthesis of compound 208 from compound 204 via compounds 205,206, and 207 according to steps a) to d).

FIG. 9: Synthesis of compound 212 from compound 209 via compounds 210and 211 according to steps a) to c).

FIG. 10: Synthesis of the pentasaccharide 203, via compounds 214, 212,215, 216, 217, 218, and 208 according to steps a) to f).

FIG. 11: Retrosynthetic analysis of the target decasaccharideD′A′B′(E′)C′DAB(E)C 301 according to various routes (a), (b) and (c).Route (a): involving synthons 306 to 310; Route (b): involving synthons311 to 313.

FIG. 12: Synthesis of the pentasaccharides 302, 303, 304 according tosteps a) to e) or f) and involving notably a coupling with atrisaccharide 309 or 310 (see FIG. 11).

FIG. 13: Synthesis of the pentasaccharide 313 from monosaccharide 314via compounds 321, 322, 323, 316, and 324-329 according to steps a) toh).

FIG. 14: Synthesis of the tetrasaccharides 338, 339, 340, 341 accordingto steps a) to e) from compound 323 via compounds 311, 332, 333, 334,335, and 336 according to steps a) or b), c) to e).

FIG. 15: Synthesis of the pentasaccharide 346 according to steps a) tof), from compound 311, via compounds 321, 342, 310, 343, 344, 340, 304,and 345 according to steps a) to f).

FIG. 16: Synthesis of the decasaccharide D′A′B′(E′)C′DAB(E)C 301 fromcompound 302 via compounds 347, 348, 346, 349, 350, 351, and 352according to steps a) to g).

FIG. 17: Retrosynthetic analysis of the target conjugate 401. Peptidedisclosed as SEQ ID NO: 40.

FIG. 18: Synthesis of the hexasaccharide 402 according to steps e) to l)from compound 407 via compounds 408, 406, 405, 409, 410, and 411.

FIG. 19: Retrosynthetic analysis of the target conjugates 501, 502, 503involving the coupling of synthons 504, 505, or 506 with 507 and thenwith 508 via a reaction with SAMA-Pfp.

FIG. 20: Synthesis of the aminoethyl ECD building block 518 fromcompounds 509 and 510, via compounds 511, 504, 512, 513, 514, 507, 515,516, and 517 according to steps a) to h).

FIG. 21: Synthesis of the aminoethyl tetrasaccharide 525 from compound511, via compounds 519, 520, 521, 522, 505, 507, 523, and 524 accordingto steps a) to g).

FIG. 22: Synthesis of the aminoethyl pentasaccharide 537 from thecompound 533, via compounds 534, 506, 507, 535, and 536 according tosteps f), c), g), h) and i), the compound 533 being obtained either fromcompound 526 via 527-530 according to steps a) to d) or from 519 via 532and 527 according to steps e) and d).

FIG. 23: Synthesis of the conjugates 501, 502, 503 from compounds 518,525, or 537 via compounds 538, 539, 540 and 508 according to steps a) toc).

FIG. 24: Retrosynthetic analysis of the target conjugates 601,602.Peptide disclosed as SEQ ID NO: 40.

FIG. 25: Synthesis of the pentasaccharides 606 from compound 609according to step a) and synthesis of 607 from compound 609 viacompounds 610-615 according to steps b) to h).

FIG. 26: Synthesis of the decasaccharide 620 according to steps a) to f)from compounds 607 and 608, and via compounds 616, 617, 606, 618, 619,and 603.

FIG. 27: Synthesis of the pentadecasaccharide 625 according to steps a)to e) from compounds 617 and 607, and via compounds 621, 622, and 606.

FIG. 28: Synthesis of the conjugates 701 to 713 from coupling of 701 to706 with 707, which is:

FIG. 28 bis: Retrosynthesis of the conjugate 801.

FIG. 29 illustrates the structure of the repeating units of the O—SP ofS. flexneri serotype 2a.

FIG. 30 illustrates the protection conferred by immune serum specificfor S. flexneri 2a LPS intranasally administered prior to i.n.challenge.

A. Serum IgG subclasses elicited in mice upon i.p. immunization withkilled S. flexneri 2a bacteria. represents the mean value of theantibody titer (n=10 mice).

B. Protection assessed by reduction of lung-bacterial load in micereceiving anti-S. flexneri 2a LPS immune serum raised upon i.p.immunization, 1 h prior to i.n. challenge with a sublethal dose of S.flexneri 2a bacteria. a, b, c, correspond to immune sera exhibiting ananti-S. flexneri 2a LPS IgG antibody titer of 1/4,000, 1/16,000 and1/64,000, respectively. Standard deviation is indicated (n=10 mice pergroup).

FIG. 31 illustrates the protection conferred by different subclasses ofmIgG specific for S. flexneri 2a serotype determinants. A: micereceiving intranasally 20 μg and 2 μg of purified mIgG (F22, D15, A2, E4or C1), respectively, 1 h prior to i.n. challenge with a sublethal doseof S. flexneri 2a bacteria. Lung-bacterial load was expressed usingarbitrary units with 100 corresponding to the bacterial count in lungsof control mice. Standard deviations are represented (n=10 mice pergroup; 3 independent experiments). B: Histopathological study of mouselungs. Upper row: control mice. Lower row: mice receiving mIgG. HEstaining: a and d magnification ×40; b and e magnification ×100.Immunostaining using an anti-LPS antibody specific for S. flexneriserotype 2a: c and f magnification ×100.

FIG. 32 illustrates the serotype-specific protection conferred by theanti-O—SP mIgGs. A: Mice were receiving i.n. 20 μg of each of thepurified mIgG, C20 and C1-7, 1 h prior to i.n. challenge with asublethal dose of S. flexneri serotype 2a (A) or serotype 5a (B)bacteria. Lung-bacterial load was expressed using arbitrary units with100 corresponding to the bacterial count in lungs of control mice.Standard deviations are represented (n=10 mice per group; 3 independentexperiments). B: Histopathological study of mouse lungs. a and b: micereceiving mIgGC20 specific for S. flexneri serotype 5a and challengedwith S. flexneri serotype 2a and 5a, respectively. c and d: micereceiving mIgGC1-7 specific for S. flexneri 2a prior to challenge withS. flexneri serotype 2a and 5a, respectively. HE staining, magnification×100.

FIG. 33 illustrates the protection conferred by mIgG specific for S.flexneri IpaB or IpaC invasins. Mice were receiving i.n. 20 μg of eachof the purified mIgG, H4, H16, J22, K24, and C20, 1 h prior to i.n.challenge with a sublethal dose of S. flexneri serotype 5a.Lung-bacterial load was expressed using arbitrary units with 100corresponding to the bacterial count in lungs of control mice. Standarddeviations are represented (n=10 mice per group).

FIG. 34 illustrates the protection conferred by oligosaccharides-tetanustoxoid conjugates in the mouse model of pulmonary infection. For eachmice tested the antiLPS 2a antibody titer before the challenge (verticalaxis) is indicated as a function of the bacteria load 24 hours after thechallenge with tetra- (FIG. 34 A), penta- (FIG. 34 B), hexa-35 (FIG. 34C), deca- (FIG. 34 D), pentadecasaccharide (FIG. 34 E) and LPS (FIG. 34F) conjugates (horizontal axis).

I Synthesis of Oligosaccharides, Polysaccharides and ConjugatesAccording to the Invention

General Methods. Melting points were determined in capillary tubes withan electrothermal apparatus and are uncorrected. Optical rotations weremeasured for CHCl₃ solutions at 25° C., expect where indicatedotherwise. TLC on precoated slides of Silica Gel 60 F₂₅₄ (Merck) wasperformed with solvent mixtures of appropriately adjusted polarity.Detection was effected when applicable, with UV light, and/or bycharring with orcinol (35 mM) in 4N aq H₂SO₄. Preparative chromatographywas performed by elution from columns of Silica Gel 60 (particle size0.040-0.063 mm). RP-HPLC (215 nm or 230 nm) used Kromasil 5 μm C18 100 Å4.6×250 mm, analytical column (1 mL·min⁻¹). NMR spectra were recorded at20° C. on a Brucker Avance 400 spectrometer (400 MHz for ¹H, 100 MHz for¹³C) at 20° C. Unless indicated otherwise, NMR spectra were run forsolutions in CDCl₃ using TMS (0.00 ppm for both ¹H and ¹³C) as anexternal reference. Dioxane (67.4 ppm for ¹³C) andtrimethylsilyl-3-propionic acid sodium salt (0.00 ppm for ¹H) were usedas external references for solutions in D₂O. Proton-signal assignmentswere made by first-order analysis of the spectra, as well as analysis of2D ¹H—¹H correlation maps (COSY) and selective TOCSY experiments. In theNMR spectra, of the two magnetically non-equivalent geminal protons atC-6, the one resonating at lower field is denoted H-6a and the one athigher field is denoted H-6b. The ¹³C NMR assignments were supported by2D ¹³C—¹H correlations maps (HETCOR). Interchangeable assignments in the¹³C NMR spectra are marked with an asterisk in listing of signalassignments. Sugar residues in oligosaccharides are serially letteredaccording to the lettering of the repeating unit of the O—SP areidentified by a subscript in listing of signal assignments. Lowresolution mass spectra were obtained by either chemical ionisation(CI-MS) using NH₃ as the ionising gas, by electrospray mass spectrometry(ES-MS), by fast atom bombardment mass spectrometry (FAB-MS) recorded inthe positive-ion mode using dithioerythridol/dithio-L-threitol (4:1,Magic Bullet) as the matrix in the presence of NaI, and Xenon as thegas. HRMS were obtained by Matrix Assisted Laser Desorption Ionisation(MALDI).

Abréviations

TCA: trichloroacetimidate

EtOAc: Ethyl acetate

1,2-DCE: 1,2-dichloroethane

DCM: Dichloromethane

THF: Tetrahydrofuran

DMF: N,N-dimethyl formamide

rt: room temperature

A—Synthesis of the Methyl Glycosides of a Tetra- and Two PentasaccharideFragments of the O-Specific Polysaccharide of Shigella flexneri Serotype2a:

Appropriate solvents for chromatography consisted of A,dichloromethane-methanol; B, cyclohexane-ethyl acetate, C,cyclohexane-acetone, D, water-acetonitrile, E,iso-propanol-ammonia-water; F, 0.01 M aq TFA-acetonitrile.

Methyl (3,4-di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl)(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (108). Activated powered 4 Åmolecular sieves (200 mg) was added to a solution of alcohol (V.Pozsgay, J.-R. Brisson, H. J. Jennings, Can. J. Chem. 1987, 65,2764-2769) 104 (60 mg, 167 μmol) and trichloroacetimidate donor 120 (113mg, 0.2 mmol) in dry Et₂O (2 mL) and the solution was stirred at rt for30 min then cooled to −40° C. TMSOTf (9 μL, 50 μmol) was added and themixture was stirred for 1 h at −30° C., then for 2 h while the bathtemperature was coming back to rt. TLC (solvent B, 4:1) showed thepresence of less polar product than 104. The mixture was neutralized byaddition of Et₃N, and filtered on a pad of Celite. Concentration of thefiltrate and column chromatography of the residue (solvent B, 4:1) gave86 mg of 108 as a colourless oil (67%). [α]_(D)-13.6 (c 1.0); ¹H NMRδ7.42-7.32 (m, 20H, Ph), 5.64 (dd, 1H, J_(1,2)=1.9, J_(2,3)=3.2 Hz,H-2_(A)), 5.07 (d, 1H, H-1_(A)), 4.98-4.93 (m, 2H, OCH₂), 4.83-4.61 (m,6H, OCH₂), 4.64 (bs, 1H, H-1_(B)), 4.18 (d, 1H, J=15.2 Hz, CH₂Cl), 4.13(d, 1H, OCH₂Cl), 3.90 (dd, 1H, J_(3,4)=9.3 Hz, H-3_(B)), 3.89 (m, 1H,partially overlapped, J_(5,6)=6.3 Hz, H-5_(A)), 3.73 (dq, 1H,J_(4,5)=9.5, J_(5,6)=6.2 Hz, H-5_(B)), 3.48 (pt, 1H, J_(3,4)=9.4 Hz,H-4_(B)), 3.45 (pt, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-4_(A)), 3.36 (s, 3H,OCH₃), 1.37 (d, 3H, H-6_(A)), 1.35 (d, 3H, H-6_(B)); ¹³C NMR δ 165.5(CO), 137.4-126.4 (Ph), 100.2 (C-1_(A)), 99.2 (C-1_(B)), 80.4, 80.3,80.2 (2C, C-4_(A), 4_(B), 3_(B)), 77.9 (C-3_(A)), 75.8, 75.7 (2C, OCH₂),74.8 (C-2_(B)), 72.6, 72.5 (2C, OCH₂), 71.2 (C-2_(A)), 68.7 (C-5_(A)),68.2 (C-5_(B)), 55.0 (OCH₃), 41.4 (CH₂Cl), 18.4 (2C, C-6_(A), 6_(B)).FABMS for C₄₃H₄₉ClNO₁₀ (M, 760.3) m/z 783.3 [M+Na]⁺. Anal. Calcd forC₄₃H₄₉ClNO₁₀: C, 67.84; H, 6.49%. Found: C, 68.03; H, 7.02.

Methyl(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(107). Activated powered 4 Å molecular sieves was added to a solution ofalcohol 104 (322 mg, 0.90 mmol) and trichloroacetimidate donor (J. C.Castro-Palomino, M. H. Rensoli, V. Verez Bencomo, J. Carbohydr. Chem.1996, 15, 137-146) 105 (573 mg, 1.08 mmol) in dry Et₂O (9 mL) and thesolution was stirred at rt for 30 min then cooled to −35° C. TMSOTf (48μL, 266 μmol) was added and the mixture was stirred for 4 h, while thebath temperature was coming back to rt. TLC (solvent B, 23:2) showedthat only little starting material remained and the mixture wasneutralized by addition of Et₃N, and filtered on a pad of Celite.Concentration of the filtrate and column chromatography of the residue(solvent B, 9:1) gave 647 mg of slightly contaminated 106. The later(626 mg) was dissolved in a mixture of CH₂Cl₂ (2 mL) and MeOH (5 mL) and1M methanolic sodium methoxide (300 μL) was added. The mixture wasstirred overnight, neutralized with Amberlite IR 120 (H⁺), filtered andconcentrated. Chromatography of the residue (solvent G, 89:11) gavesyrupy 107 (554 mg, 91% from 104). Analytical data were as described.(V. Pozsgay, J.-R. Brisson, H. J. Jennings, Can. J. Chem. 1987, 65,2764-2769)

Methyl(3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophtalimido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(110). A solution of disaccharide 107 (179 mg, 0.26 mmol) andtrichloroacetimidate donor (J. C. Castro-Palomino, R. R. Schmidt,Tetrahedron Lett. 1995, 36, 5343-5346) 109 (436 mg, 0.60 mmol) in dryCH₃CN (9 mL) was stirred at rt for 30 min in the presence of activated 4Å molecular sieves (1.2 g). Tin(II) trifluoromethanesulfonate [Sn(OTf)₂](75 mg, 180 μmol) was added and the mixture was stirred at rt for 4 h,then neutralized with Et₃N. Filtration on a pad of Celite, concentrationof the filtrate and column chromatography of the residue (solvent B,87:13) gave 110 (324 mg) as a slightly contaminated white foam (72% asestimated from the ¹H NMR spectrum). An analytical sample had[α]_(D)+23.3 (c 1.0); ¹H NMR δ 7.43-7.17 (m, 20H, Ph), 5.92 (d, 1H,J=9.2, J=10.5 Hz, H-3_(D)), 5.24 (d, 1H, J_(1,2)=8.4 Hz, H-1_(D)), 5.14(dd, 1H, J=9.7, J=9.4 Hz, H-4_(D)), 5.00 (bs, 1H, H-1_(A)), 4.79 (d, 1H,J=10.8 Hz, OCH₂), 4.65 (s, 2H, OCH₂), 4.55 (d, 1H, J=11.2 Hz, OCH₂),4.53 (bs, 1H, H-1_(B)), 4.46-4.36 (m, 3H, H-2_(D), OCH₂), 4.28 (d, 1H,J=12.4 Hz, OCH₂), 4.26 (d, 1H, J=10.6 Hz, OCH₂), 4.06 (dd, 1H,J_(6a,6b)=12.5, J_(5,6a)=6.8 Hz, H-6a_(D)), 3.91 (bs, 1H, H-2_(B)),3.85-3.69 (m, 5H, H-2_(A), H-3_(B), 3_(A), 6b_(D), 5_(A)*), 3.59 (dq,1H, J_(4,5)=9.4, J_(5,6)=6.2 Hz, H-5_(B)*), 3.40 (m, 1H, H-5_(D)), 3.27(s, 3H, OCH₃), 3.18 (m, 2H, H-4_(A), 4_(B)), 2.03, 2.01, 1.94 (3s, 9H,C(O)CH₃), 1.27, 1.25 (2d, 6H, H-6_(A), 6_(B)); ¹³C NMR δ 170.5, 170.4,170.3, 163.8, 162.6 (5C, CO), 140.3-128.0 (Ph), 101.1 (C-1_(A)), 100.0(C-1_(D)), 99.8 (C-1_(B)), 80.7 (2C, C-4_(A), 4_(B)), 79.7 (C-2_(A)),78.9 (C-3_(B)), 78.1 (C-3_(A)), 76.2 (C-2_(B)), 75.3, 75.2, 72.7, 71.4(4C, OCH₂), 71.3 (C-5_(D)), 70.1 (C-3_(D)), 68.5 (C-5_(A)*), 68.4(C-4_(D)), 67.4 (C-5_(B)*), 61.3 (C-6_(D)), 55.4 (C-2_(D)), 54.6 (OCH₃),20.7, 20.6 (3C, C(O)CH₃), 18.0, 17.7 (2C, C-6_(A), 6_(B)). FABMS forC₆₁H₆₃Cl₄NO₁₈ (M, 1237.3) m/z 1259.9 [M+Na]⁺. Anal. Calcd forC₆₁H₆₃Cl₄NO₁₈.H₂O: C, 58.24; H, 5.21; N, 1.11%. Found: C, 58.21; H,4.91; N, 1.01%.

Methyl(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(111). A solution of disaccharide 107 (179 mg, 0.26 mmol) andtrichloroacetimidate donor 109 (436 mg, 0.60 mmol) in dry CH₃CN (9 mL)was stirred at rt for 30 min in the presence of activated 4 Å molecularsieves (1.2 g). Tin(II) trifluoromethanesulfonate [Sn(OTf)₂] (75 mg, 180μmol) was added and the mixture was stirred at rt for 4 h, thenneutralized with Et₃N. Filtration on a pad of Celite, concentration ofthe filtrate and column chromatography of the residue (solvent B, 87:13)gave 110 (324 mg) as a slightly contaminated product. The latter wassolubilized in dry ethanol (13 mL) and diethylamine (200 μL, 3.0 mmol)was added and the mixture was stirred overnight at 60° C. The mixturewas cooled to rt and acetic anhydride (1.0 mL, 10.6 mmol) was added andthe mixture was stirred at this temperature for 2 h. The suspension wasfiltered and volatiles were evaporated and coevaporated repeatedly withtoluene and cyclohexane. The crude residue was taken up in a minimum ofCH₂Cl₂ and MeOH (10 mL). 1N methanolic sodium methoxide was added untilthe pH was 10 and the solution was stirred overnight at rt, neutralizedwith IR 120 (H⁺), filtered and concentrated. Chromatography of theresidue (solvent A, 24:1) gave foamy 111 (135 mg, 51% from 107).[α]_(D)−15.0 (c 1.0); ¹H NMR δ 7.44-7.28 (m, 20H, Ph), 8.88 (bs, 1H,NH_(D)), 5.28 (bs, 1H, H-1_(A)), 4.93-4.61 (m, 8H, OCH₂), 4.59 (s, 1H,J_(1,2)=1.3 Hz, H-1_(B)), 4.41 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 4.06(m, 2H, H-2_(A), 2_(B)), 4.00 (dd, 1H, J_(2,3)=3.3, J_(3,4)=9.4 Hz,H-3_(A)), 3.86 (dd, 1H, J_(2,3)=2.9, J_(3,4)=9.4 Hz, H-3_(B)), 3.79 (dq,1H, J_(4,5)=9.4, J_(5,6)=6.2 Hz, H-5_(A)*), 3.67 (m, 2H, H-5_(B)*,6a_(D)), 3.51 (m, 1H, H-2_(D)), 3.49-3.38 (m, 6H, H-6b_(D), 4_(D),3_(D), 4_(B), 4_(A)), 3.31 (s, 3H, OCH₃), 3.29 (m, 1H, H-5_(D)). 1.55(s, 3H, C(O)CH₃), 1.35 (d, 6H, H-6_(A), 6_(B)); ¹³C NMR δ 173.6 (CO),138.5-127.6 (Ph), 103.2 (C-1_(D)), 100.2 (C-1_(A)), 99.9 (C-1_(B)),81.3, 80.7 (2C, C-4_(A), 4_(B)), 79.9 (2C, C-3_(A), 3_(B)), 79.0(C-2_(A)), 77.2 (C-3_(D)), 75.8 (C-5_(D)), 75.7, 75.2, 74.6 (3C, OCH₂),73.4 (C-2_(B)), 72.3 (OCH₂), 71.8 (C-4_(D)), 68.2, 67.7 (C-5_(A),5_(B)), 62.5 (C-6_(D)), 58.9 (C-2_(D)), 54.6 (OCH₃), 22.3 (C(O)CH₃),17.9, 17.7 (2C, C-6_(A), 6_(B)). FABMS for C₄₉H₆₁NO₁₄ (M, 887.44) m/z910.1 [M+Na]⁺. Anal. Calcd for C₄₉H₆₁NO₁₄.H₂O: C, 64.96; H, 7.01; N,1.55%. Found: C, 65.19; H, 6.83; N, 1.51%.

Methyl(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(112). 2,2-dimethoxypropane (4.9 mL, 39.8 mmol) and para-toluenesulfonicacid (18 mg, 95 μmol) were added to a solution of the triol 111 (964 mg,1.09 mmol) in acetone (3 mL) and the mixture was stirred at rt for 1 h.Et₃N was added, and volatiles were evaporated. Column chromatography ofthe residue (solvent A, 99:1) gave the acceptor 112 as a white solid(969 mg, 96%) which could be crystallized from AcOEt:iPr₂O; mp 164-165°C. [α]_(D)−25.9 (c 1.0); ¹H NMR δ 7.45-7.31 (m, 20H, Ph), 6.98 (d, 1H,J_(NH,2)=2.4 Hz, NH), 6.37 (bs, 1H, OH), 5.07 (d, 1H, J_(1,2)=1.9 Hz,H-1_(A)), 4.90 (d, 1H, J=10.8 Hz, OCH₂), 4.85 (d, 1H, J=10.1 Hz, OCH₂),4.84 (d, 1H, J=10.8 Hz, OCH₂), 4.76 (d, 1H, OCH₂), 4.69 (d, 1H, OCH₂),4.68 (s, 2H, OCH₂), 4.65 (d, 1H, OCH₂), 4.61 (d, 1H, J_(1,2)=1.6 Hz,H-1_(B)), 4.48 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 4.09 (dd, 1H, H-2_(A)),4.01 (dd, 1H, J_(2,3)=3.2, J_(3,4)=9.4 Hz, H-3_(A)), 3.91 (dd, 1H,H-2_(B)), 3.89-3.84 (m, 2H, J_(5,6)=6.3, J_(4,5)=9.4, J_(2′,3′)=3.3,J_(3′,4′)=9.4 Hz, H-5_(A), 3_(B)), 3.68 (dq, partially overlapped,J_(5,6)=6.2, J_(4,5)=9.5 Hz, H-5_(B)), 3.66-3.58 (m, 5H, H-6a_(D),6b_(D), 2_(D), 3_(D), 4_(D)), 3.44 (pt, 1H, H-4_(A)), 3.41 (pt, 1H,H-4_(B)), 3.32 (s, 3H, OCH₃), 3.16 (m, 1H, H-5_(D)), 1.60 (s, 3H,C(O)CH₃), 1.54, 1.48 (2s, 6H, C(CH₃)₂), 1.35 (d, 6H, H-6_(A), 6_(B));¹³C NMR δ 173.9 (CO), 138.8-128.0 (Ph), 103.7 (C-1_(D)), 101.3(C-1_(A)), 100.3 (C(CH₃)₂), 100.2 (C-1_(B)), 81.9 (C-4_(A)), 80.8(C-4_(B)), 80.5 (C-3_(A)), 79.7 (C-3_(B)), 79.4 (C-2_(A)), 76.2 (OCH₂),76.0 (C-2_(B)), 75.6, 75.1 (2C, OCH₂), 74.7 (C-4_(D)), 74.4 (C-3_(D)),72.6 (OCH₂), 68.6 (C-5_(A)), 68.0, 67.9 (2C, C-5_(B), 5_(D)), 62.2(C-6_(D)), 60.6 (C-2_(D)), 55.1 (OCH₃), 29.5 (C(CH₃)₂), 22.7 (C(O)CH₃),19.4 (C(CH₃)₂), 18.5, 18.2 (2C, C-6_(A), 6_(B)). FABMS for C₅₂H₆₅NO₁₄(M, 927.44) m/z 950.1 [M+Na]⁺. Anal. Calcd for C₅₂H₆₅NO₁₄: C, 67.30; H,7.06; N, 1.51%. Found: C, 67.12; H, 6.98; N, 1.44%.

Methyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(115). Activated powdered 4 Å molecular sieves were added to a solutionof the trisaccharide acceptor 112 (202 mg, 0.22 mmol) and thedisaccharide donor 114 (263 mg, 0.25 mmol) in anhydrous CH₂Cl₂ (5 mL)and the suspension was stirred for 30 min at −15° C. TfOH (7 μL, 34μmol) was added and the mixture was stirred for 2 h while the bathtemperature was slowly coming back to 10° C. TLC (solvent D, 49:1)showed that no 112 remained. Et₃N was added and after 30 min, thesuspension was filtered through a pad of Celite. Concentration of thefiltrate and chromatography of the residue (solvent B, 9:1→17:5) gavethe fully protected pentasaccharide 115 (330 mg, 84%) as a white foam;[α]_(D)+63.3 (c 1.0); ¹H NMR δ 8.07-6.96 (m, 50H, Ph), 5.82 (d, 1H,J_(NH,2)=7.4 Hz, NH), 5.63 (dd, 1H, J_(2,3)=3.5, J_(3,4)=9.5 Hz,H-3_(C)), 5.43 (dd, 1H, J_(1,2)=1.6 Hz, H-2_(C)), 5.09 (bs, 1H,H-1_(A)), 5.02 (d, 1H, J_(1,2)=3.4 Hz, H-1_(E)), 4.99 (d, 1H,J_(1,2)=8.3 Hz, H-1_(D)), 4.95 (d, 1H, J_(1,2)=1.1 Hz, H-1_(C)),4.94-4.63 (m, 13H, OCH₂), 4.63 (s, 1H, H-1_(B)), 4.37 (d, 1H, J=11.0 Hz,OCH₂), 4.29 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2 Hz, H-5_(C)), 4.25 (d, 1H,J=9.5 Hz, OCH₂), 4.23 (pt, 1H, J_(3,4)=J_(4,5)=9.5 Hz, H-3_(D)), 4.01(m, 1H, H-2_(A)), 3.97-3.86 (m, 5H, H-3_(A), 2_(B), 3_(E), 4_(C), OCH₂),3.82 (m, 1H, H-3_(B), 5_(A)), 3.71-3.57 (m, 7H, H-5_(D), 4_(E), 5_(B),4_(D), 6a _(D), 6b_(D)), 3.54-3.41 (m, 3H, H-2_(E), 4_(A), 2_(D))3.38-3.31 (m, 2H, H-4_(B), 6a_(E)), 3.31 (s, 3H, OCH₃), 3.17 (m, 1H,H-5_(D)), 3.08 (d, 1H, J_(6a,6b)=10.1 Hz, H-6b_(E)), 1.84 (s, 3H,C(O)CH₃), 1.46 (s, 3H, C(CH₃)₂), 1.45 (d, 3H, J_(5,6)=5.9 Hz, H-6_(C)),1.35 (m, 6H, J_(5,6)=5.9 Hz, H-6_(A), C(CH₃)₂), 1.31 (d, 3H, J_(5,6)=6.2Hz, H-6_(B)); ¹³C NMR δ 171.7, 165.9, 165.8 (3C, CO), 138.9-127.9 (Ph),102.3 (C-1_(D), J=167 Hz), 101.5 (C-1_(A), J=170 Hz), 100.3 (C-1_(B),J=170 Hz), 99.8 (C(CH₃)₂), 99.6 (C-1_(E), J=172 Hz), 98.2 (C-1_(C),J=172 Hz), 82.0 (C-3_(E)), 81.2, 80.9, 80.7 (3C, C-4_(A), 4_(B), 2_(E)),80.0, 79.7, 79.3 (3C, C-3_(B), 3_(A), 4_(C)), 78.1, 77.8, 77.4 (3C,C-2_(A), 4_(E), 3_(D)), 75.9, 75.8, 75.6 (3C, OCH₂), 75.5 (C-2_(B)),75.0, 74.4, 73.7 (3C, OCH₂), 73.2 (2C, C-4_(D), OCH₂), 72.2 (OCH₂),71.7, 71.6 (3C, C-2_(C), 3_(C), 5_(E)), 68.8 (C-5_(B)), 68.0 (C-6_(E)),68.0 (2C, C-5_(A), 5_(B)), 67.6 (C-5_(D)), 62.5 (C-6_(D)), 58.9(C-2_(D)), 55.0 (OCH₃), 29.5 (C(CH₃)₂), 23.8 (C(O)CH₃), 19.8 (C(CH₃)₂),18.6 (C-6_(C)), 18.5 (C-6_(A)), 18.3 (C-6_(B)). FAB-MS for C₁₀₆H₁₁₇NO₂₅(M, 1803.79) m/z 1826.4 [M+H]⁺. Anal. Calcd for C₁₀₆H₁₁₇NO₂₅.H₂O: C,69.83; H, 6.58; N, 0.77%. Found: C, 69.86; H, 6.33; N, 0.71%.

Methyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(116). 90% aq TFA (750 μL) was added at 0° C. to a solution of the fullyprotected 115 (588 mg, 326 μmol) in CH₂Cl₂ (6.7 mL) and the mixture wasstirred at this temperature for 1 h. TLC (solvent B, 1.5:1) showed thatno 115 remained. Volatiles were evaporated by repeated addition oftoluene. Chromatography of the residue (solvent B, 4:1→1:1) gave 116(544 mg, 95%) as a white foam; [α]_(D)+58.8 (c 1.0); ¹H NMR δ 8.06-7.06(m, 50H, Ph), 5.82 (d, 1H, J_(NH,2)=7.1 Hz, NH), 5.65 (dd, 1H,J_(2,3)=3.8, J_(3,4)=9.0 Hz, H-3_(C)), 5.53 (m, 1H, H-2_(C)), 5.34 (bs,1H, H-1_(A)), 5.04 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 5.00 (m, 2H,H-1_(C), 1_(E)), 4.97-4.63 (m, 13H, OCH₂), 4.48 (bs, 1H, H-1_(B)), 4.40(d, 1H, J=8.4 Hz, OCH₂), 4.29 (d, 1H, J=8.0 Hz, OCH₂), 4.28-4.21 (m, 2H,H-3_(D), 5_(C)), 4.10 (m, 1H, H-2_(B)), 4.04 (m, 1H, H-2_(A)), 3.99 (d,1H, OCH₂), 3.95-3.89 (m, 3H, H-3_(A), 3_(E), 4_(C)), 3.87 (dd, 1H,J_(2,3)=2.7, J_(3,4)=9.7 Hz, H-3_(B)), 3.81-3.64 (m, 5H, H-5_(E), 5_(A),6a_(D), 4_(E), 5_(B)), 3.54 (dd, 1H, J_(1,2)=3.2, J_(2,3)=9.7 Hz,H-2_(E)), 3.51 (pt, 1H, J_(3,4)=J_(4,5)=9.5 Hz, H-4_(A)), 3.45-3.37 (m,4H, H-4_(B), 4_(D), 6a_(E), 2_(D)), 3.33 (m, 5H, H-5_(D), 6b_(D), OCH₃),3.12 (d, 1H, J_(6a,6b)=10.6 Hz, H-6b_(E)), 2.28 (bs, 1H, OH), 1.97 (bs,1H, OH), 1.84 (s, 3H, C(O)CH₃), 1.54 (d, 3H, J_(5,6)=6.1 Hz, H-6_(C)),1.37 (m, 6H, H-6_(B), 6_(A)); ¹³C NMR δ 171.5, 165.8, 165.6 (3C, CO),138.8-127.9 (Ph), 101.6 (C-1_(D)), 100.8 (C-1_(A)), 100.5 (C-1_(B)),100.1 (C-1_(E)*), 99.9 (C-1_(C)*), 84.9 (C-3_(D)), 82.1 (C-3_(E)), 80.9,80.7, 80.6, 80.5 (4C, C-4_(B), 3_(B), 4_(A), 2_(E)), 79.7 (C-4_(C)),79.3 (C-3_(A)), 77.8 (2C, C-2_(A), 4_(E)), 76.0, 75.9 (2C, OCH₂), 75.8(C-5_(D)), 75.6, 75.1, 74.6, 73.7, 73.1 (5C, OCH₂), 72.8 (C-2_(B)), 72.6(OCH₂), 71.8 (C-5_(E)), 71.6 (C-4_(D)), 71.3 (C-3_(C)), 71.1 (C-2_(C)),69.4 (C-5_(C)), 68.8 (C-5_(A)), 68.3 (C-5_(B)), 68.1 (C-6_(E)), 63.0(C-6_(D)), 57.6 (C-2_(D)), 55.0 (OCH₃), 23.8 (C(O)CH₃), 18.8 (C-6_(C)),18.6, 18.5 (2C, C-6_(A), 6_(B)). FAB-MS for C₁₀₃H₁₁₃NO₂₅ (M, 1763.76)m/z 1786.2 [M+H]⁺. Anal. Calcd for C₁₀₃H₁₁₃NO₂₅.2H₂O: C, 68.69; H, 6.55;N, 0.78%. Found: C, 68.74; H, 6.45; N, 0.65%.

Methyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-α-L-rhamnopyranosyl-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(117). 1M Methanolic sodium methoxide was added to a solution of 116(277 mg, 157 μmol) in a 1:1 mixture of CH₂Cl₂ and MeOH (6 mL) until thepH was 10. The mixture was stirred overnight at rt and neutralized withAmberlite IR-120 (H⁺). The crude material was chromatographed (solventA, 49:1) to give 117 (211 mg, 86%) as a white foam; [α]_(D)+23.8 (c1.0); ¹H NMR δ 7.33-7.16 (m, 40H, Ph), 5.34 (d, 1H, J_(NH,2)=7.6 Hz,NH), 5.18 (bs, 1H, H-1_(A)), 4.79 (d, partially overlapped, 1H,H-1_(E)), 4.67 (bs, 1H, H-1_(C)), 4.50 (d, partially overlapped, 1H,H-1_(D)), 4.49 (bs, 1H, H-1_(B)), 4.88-4.33 (m, 16H, OCH₂), 3.98-3.81(m, 6H, H-2_(A), 2_(B), 5_(E), 3_(A), 3_(E), 5_(B)*), 3.77-3.70 (m, 3H,H-3_(B), 2_(C), 5_(C)*), 3.65 (dq, 1H, J_(4,5)=9.4, J_(5,6)=6.2 Hz,H-5_(A)*), 3.62-3.51 (m, 4H, H-2_(D), 6a_(D), 6a_(E), 6b_(E)), 3.48-3.27(m, 7H, H-2_(E), 4_(E), 3_(D), 4_(A), 4_(B), 3_(C), 4_(C)), 3.23-3.12(m, 6H, H-4_(D), 6b_(D), 5_(D), OCH₃), 2.76 (bs, 1H, OH), 1.72 (bs, 3H,OH), 1.65 (s, 3H, NHAc), 1.32, 1.25 (2d, 9H, H-6_(C), 6_(B), 6_(A)); ¹³CNMR δ 170.6 (CO), 138.5-128.0 (Ph), 103.0 (C-1_(D)), 101.8 (C-1_(C)),100.7 (C-1_(A)), 100.4 (C-1_(B)), 99.6 (C-1_(E)), 87.3 (C-3_(D)), 85.(C-4_(C)*), 82.0 (C-3_(E)), 81.2, 80.7, 80.5, 80.2, 797, 78.1, 77.9 (7C,C-2_(B), 3_(A), 3_(B), 4_(A), 4_(B), 2_(E), 4_(E)), 76.2 (C-5_(D)),76.1, 75.9, 75.6, 75.4, 74.0, 73.9, 73.6 (7C, OCH₂), 73.0 (C-2_(A)),72.8 (OCH₂), 71.7, 71.2, 71.1, 69.8 (4C, C-4_(D), 5_(E), 2_(C), 3_(C)),68.8, 68.2 (3C, C-5_(A), 5_(B), 5_(C)), 63.1 (C-6_(D)), 55.6 (C-2_(D)),55.0 (OCH₃), 23.7 (C(O)CH₃), 18.6, 18.3, 18.1 (3C, C-6_(A), 6_(B),6_(C)). FAB-MS for C₈₉H₁₀₅NO₂₃ (M, 1555.71) m/z 1578.2 [M+H]⁺. Anal.Calcd for C₈₉H₁₀₅NO₂₃: C, 68.66; H, 6.80; N, 0.90%. Found: C, 68.41; H,6.78; N, 0.61%.

Methylα-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranoside(101). The benzylated tetrasaccharide 117 (352 mg, 226 μmol) wasdissolved in a mixture of ethanol (14 mL) and AcOH (1 mL), treated with10% Pd—C catalyst (200 mg), and the suspension was stirred for 5 days atrt. TLC (solvent A, 1:1) showed that the starting material had beentransformed into a more polar product. The suspension was filtered on apad of Celite. The filtrate was concentrated and coevaporated repeatedlywith cyclohexane. Reverse phase chromatography of the residue (solventD, 100:0→49:1), followed by freeze-drying, gave the targettetrasaccharide 101 as an amorphous powder (153 mg, 81%). RP-HPLC gave asingle product eluting at Rt: 15.21 min (solvent F, 1:0→80:20 over 20min); [α]_(D)−3.2 (c 1.0, methanol); ¹H NMR (D₂O) δ 5.08 (d, 1H,J_(1,2)=1.2 Hz, H-1_(A)), 4.97 (d, 1H, J_(1,2)=3.9 Hz, H-1_(E)), 4.79(d, 1H, J_(1,2)=1.3 Hz, H-1_(C)), 4.69 (m, 2H, H-1_(B), 1_(D)), 4.07(dd, 1H, J_(2,3)=3.3 Hz, H-2_(A)), 4.02 (dq, 1H, J_(4,5)=9.3,J_(5,6)=6.2 Hz, H-5_(C)), 3.93 (m, 1H, H-5_(E)), 3.86 (m, 2H, H-2_(B),3_(A)), 3.82-3.73 (m, 7H, H-3_(C), 2_(D), 6a_(E), 6b_(E), 3_(B), 2_(C),6a_(D)), 3.70-3.59 (m, 4H, H-5_(A), 3_(E), 6b_(D), 5_(B)), 3.56 (pt, 1H,J_(3,4)=J_(4,5)=9.4 Hz, H-3_(D)), 3.49 (dd, 1H, J_(2,3)=9.6 Hz,H-2_(E)), 3.46-3.38 (m, 5H, H-4_(C), 4_(B), 4_(D), 5_(D), 4_(E)), 3.32(s, 3H, OCH₃), 3.24 (pt, 1H, J_(3,4)=J_(4,5)=9.6 Hz, H-4_(A)), 2.00 (s,3H, C(O)CH₃), 1.25 (d, 3H, partially overlapped, H-6_(C)), 1.23 (d, 3H,partially overlapped, H-6_(B)), 1.18 (d, 3H, J_(5,6)=6.2 Hz, H-6_(A));¹³C NMR (D₂O) δ 175.0 (CO), 102.3 (C-1_(D), J=162 Hz), 101.5 (C-1_(C),J=170 Hz), 101.3 (C-1_(A), J=173 Hz), 100.0 (C-1_(E), J=170 Hz), 99.9(C-1_(B), J=172 Hz), 81.9 (C-3_(D)), 81.4 (C-4_(C)), 79.2 (C-2_(A)),79.0 (C-2_(B)), 76.2, 73.1, 72.6, 72.2, 72.0, 71.4, 70.4, 70.0, 69.8,69.7, 69.6, 69.3, 68.9, 68.7 (14C, 3_(A), 4_(A), 5_(A), 3_(B), 4_(B),5_(B), 2_(C), 3_(C), 4_(D), 5_(D), 2_(E), 3_(E), 4_(E), 5_(E)), 68.4(C-5_(C)), 60.5 (2C, C-6_(D), 6_(E)), 56.0 (C-2_(D)), 55.3 (OCH₃), 22.6(C(O)CH₃), 17.0 (3C, C-6_(A), 6_(B), 6_(C)). HRMS (MALDI) Calcd forC₂₇H₄₇NO₁₉+Na: 858.3214. Found: 858.3206.

3,4-Di-O-benzyl-2-O-chloroacetyl-α/β-L-rhamnopyranose (128).1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (Ir(I), 25 mg) was dissolved in dry THF (5 mL) andthe resulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the colour to change toyellow. The solution was then degassed again in an argon stream. Asolution of rhamnopyranoside (P. Westerduin, P. E. der Haan, M. J. Dees,J. H. van Boom, Carbohydr. Res. 1988, 180, 195-205) 127 (3.28 g, 7.12mmol) in THF (30 mL) was degassed and added. The mixture was stirredovernight at rt, and a solution of iodine (3.6 g, 14.2 mmol) in amixture of THF (70 mL) and water (20 mL) was added. The mixture wasstirred at rt for 1 h, then concentrated. The residue was taken up inCH₂Cl₂ and washed twice with 5% aq NaHSO₄. The organic phase was driedand concentrated. The residue was purified by column chromatography(solvent B, 9:1) to give 128 (2.53 g, 85%). ¹H NMR δ 7.40-7.28 (m, 10H,Ph), 5.57 (bd, 0.2H, H-2β), 5.45 (dd, 0.8H, J_(1,2)=2.0 Hz, H-2α), 5.13(bd, 0.8H, H-1α), 4.92 (d, 1H, J=10.9 Hz, OCH₂α, OCH₂β), 4.79 (d, 0.2H,J=11.2 Hz, OCH₂β), 4.74 (d, 1H, J=11.2 Hz, OCH₂α, H-1β), 4.65 (d, 0.8H,OCH₂α), 4.64 (d, 0.2H, OCH₂β), 4.58 (d, 0.8H, OCH₂α), 4.54 (d, 0.2H,OCH₂β), 4.30 (d, 0.2H, J=15.1 Hz, CH₂Clβ), 4.26 (d, 0.2H, CH₂Clβ), 4.20(s, 1.6H, CH₂Clα), 4.08 (dd, 0.8H, J_(2,3)=3.3, J_(3,4)=9.6 Hz, H-3α),4.04 (dq, 0.8H, J_(4,5)=9.5 Hz, H-5α), 3.66 (dd, 0.2H, J_(2,3)=3.2,J_(3,4)=8.7 Hz, H-3β), 3.44 (pt, 2H, H-4α, 5β, OH-1α, 1β), 3.38 (pt,0.2H, J_(4,5)=9.5 Hz, H-4β), 1.37 (d, 0.6H, J_(5,6)=5.7 Hz, H-6β), 1.34(d, 2.4H, J_(5,6)=6.2 Hz, H-6α); ¹³C NMR δ 167.8 (COβ), 167.4 (COα),138.6-128.2 (Ph), 93.0 (C-1β), 92.4 (C-1α), 80.3 (C-4α), 80.2 (C-3β),79.6 (C-4β), 77.8 (C3α), 75.9 (OCH₂β), 75.8 (OCH₂α), 72.5 (OCH₂α), 72.3(0.4C, C-5β, OCH₂β), 71.9 (C-2-β), 71.7 (C-2α), 68.2 (C-5α), 41.3(CH₂Clα, CH₂Clβ), 18.3 (C-6α, 6β); FAB-MS for C₂₂H₂₅ClO₆ (M, 420.5) m/z443.1 [M+Na]⁺. Anal. Calcd for C₂₂H₂₅ClO₆: C, 62.78; H, 5.94%. Found: C,62.92; H, 6.11%.

3,4-Di-O-benzyl-2-O-chloroacetyl-α/β-L-rhamnopyranosyltrichloroacetimidate (120). (a) The hemiacetal 128 (700 mg, 1.66 mmol)was dissolved in CH₂Cl₂ (6 mL) and the solution was cooled to 0° C.Trichloroacetonitrile (1.7 mL) and DBU (26 μL) were added. The mixturewas stirred at rt for 2 h. Toluene was added, and co-evaporated twicefrom the residue. The crude material was purified by flashchromatography (solvent B 4:1+0.1% Et₃N) to give 120 as a white foam(687 mg, 73%, α/β:4/1).

(b) The hemiacetal 128 (858 mg, 2.04 mmol) was dissolved in CH₂Cl₂ (11mL) and freshly activated K₂CO₃ (1.1 g, 8.0 mmol) was added. Thesuspension was cooled to 0° C., and trichloroacetonitrile (1.0 mL) wasadded. The mixture was stirred vigorously at rt for 5 h. The suspensionwas filtered on a pad of Celite, and concentrated. The crude materialwas purified by flash chromatography (solvent B, 9:1+0.1% Et₃N) to give120 as a white foam (840 mg, 72%, α/β:9/1 from the ¹H NMR spectrum). ¹HNMR (α-anomer) δ 8.71 (s, 1H, NH), 7.40-7.30 (m, 10H, Ph), 6.24 (d, 1H,J_(1,2)=1.8 Hz, H-1), 5.57 (dd, 1H, H-2), 4.94 (d, 1H, J=10.8 Hz, OCH₂),4.76 (d, 1H, J=11.2 Hz, OCH₂), 4.67 (d, 1H, OCH₂), 4.62 (d, 1H, OCH₂),4.22 (s, 2H, CH₂Cl), 4.04 (dd, 1H, J_(2,3)=3.2 Hz, H-3), 3.99 (dq, 1H,J_(4,5)=9.6 Hz, H-5), 3.53 (pt, H, H-4), 1.37 (d, 3H, J_(5,6)=6.2 Hz,H-6); ¹³C NMR (α-anomer) δ 166.9 (CO), 160.4 (C═NH), 138.4-128.3 (Ph),95.2 (C-1), 91.1 (CCl₃), 79.5 (C-4), 77.6 (C-3), 76.1, 72.9 (2C, OCH₂),71.2 (C-5), 69.8 (C-2), 41.1 (CH₂Cl), 18.3 (C-6).

Allyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-2-O-benzoyl-3-O-chloroacetyl-α-L-rhamnopyranoside(122). To a solution of the known 121 (F. Segat, L. A. Mulard,Tetrahedron: Asymmetry 2002, 13, 2211-2222) (7.10 g, 8.55 mmol) in amixture of CH₂Cl₂ (40 mL) and pyridine (5 mL) at 0° C. was addedchloroacetic anhydride (3.65 g, 21.3 mmol), and the mixture was stirredat this temperature for 2 h. TLC (solvent C, 9:1) showed the completedisappearance of the starting material. MeOH (10 mL) was added, andafter 30 min, volatiles were evaporated. Column chromatography (solventB, 1:0→4:1) of the crude yellow oil afforded 122 as a colourless foam(7.34 g, 95%). [α]_(D)+47.5 (c 1.0); ¹H NMR δ 8.12-7.13 (m, 25H, Ph),5.95 (m, 1H, CH═), 5.50-5.42 (m, 2H, J_(2,3)=3.6 Hz, H-2_(C), 3_(C)),5.37 (m, 1H, ═CH₂), 5.28 (m, 1H, ═CH₂), 4.96 (d, 1H, J=11.0 Hz, OCH₂),4.93 (d, 1H, J_(1,2)=1.5 Hz, H-1_(C)), 4.90 (d, 1H, J_(1,2)=3.3 Hz,H-1_(E)), 4.87-4.81 (m, 3H, OCH₂), 4.67 (d, 1H, J=12.1 Hz, OCH₂), 4.64(d, 1H, J=12.8 Hz, OCH₂), 4.47 (d, 1H, J=10.8 Hz, OCH₂), 4.43 (d, 1H,J=12.0 Hz, OCH₂), 4.25 (m, 2H, OCH₂), 4.09 (d, 1H, J=15.5 Hz, CH₂Cl),3.99-3.93 (m, 3H, CH₂Cl, H-5_(C), 3_(C)), 3.84 (m, 1H, H-5_(E)),3.78-3.74 (m, 2H, H-6a_(E), 4_(E)), 3.70 (pt, 1H, J_(4,5)=J_(3,4)=9.3Hz, H-4_(C)), 3.58-3.54 (m, 2H, H-6b_(E), 2_(E)), 1.50 (d, 3H,J_(5,6)=6.2 Hz, H-6_(C)); ¹³C NMR δ 167.0, 166.0 (2C, CO), 139.1-128.0(Ph, All), 118.5 (All), 99.5 (C-1_(E)), 96.8 (C-1_(C)), 81.9 (C-3_(E)),81.0 (C-2_(E)), 79.7 (C-4_(C)), 77.7 (C-4_(E)), 76.0, 75.4, 74.1, 73.8(4C, OCH₂), 73.5 (C-3_(C)), 71.8 (C-5_(E)), 70.9 (C-2_(C)), 68.8 (OCH₂),68.1 (C-6_(E)), 67.7 (C-5_(C)), 41.5 (CH₂Cl), 18.6 (C-6_(C)); FAB-MS forC₅₂H₅₅O₁₂ (M, 906.5) m/z 929.3 [M+Na]⁺. Anal. Calcd for C₅₂H₅₅ClO₁₂: C,68.83; H, 6.11%. Found: C, 68.74; H, 6.19%.

(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-2-O-benzoyl-3-O-chloroacetyl-α/β-L-rhamnopyranose(123). A solution of 122 (7.21 g, 7.95 mmol) in THF (80 mL) containingactivated iridium complex (60 mg) was treated as described for thepreparation of 128. The mixture was stirred at rt for 3 h, at whichpoint a solution of iodine (4.0 g, 15.7 mmol) in a mixture of THF (90mL) and water (24 mL) was added. The mixture was stirred at rt for 30min, then concentrated. The residue was taken up in CH₂Cl₂ and washedtwice with 5% aq NaHSO₄, then with brine. The organic phase was driedand concentrated. The residue was purified by column chromatography(solvent B, 4:1) to give 123 (6.7 g, 97%) as a slightly yellow foam. ¹HNMR δ 8.10-7.09 (m, 25H, Ph), 5.47 (dd, 1H, J_(2,3)=3.5, J_(3,4)=9.3 Hz,H-3_(C)), 5.41 (bs, 1H, H-2_(C)), 5.03 (bs, 1H, H-1_(C)), 4.94 (d, 1H,J=10.9 Hz, OCH₂), 4.87 (d, 1H, J_(1,2)=3.4 Hz, H-1_(E)), 4.85 (d, 1H,OCH₂), 4.80 (m, 2H, OCH₂), 4.64 (m, 2H, OCH₂), 4.45 (d, 1H, J=10.7 Hz,OCH₂), 4.41 (d, 1H, J=12.1 Hz, OCH₂), 4.16 (dq, 1H, J_(4,5)=9.3 Hz,H-5_(C)), 4.09 (d, 1H, J=15.6 Hz, CH₂Cl), 3.96 (d, 1H, CH₂Cl), 3.93 (pt,1H, H-3_(E)), 3.83 (m, 1H, H-5_(E)), 3.77-3.68 (m, 2H, H-4_(E), 6a_(E)),3.65 (pt, 1H, H-4_(C)), 3.54 (m, 2H, H-6b_(E), 2_(E)), 1.48 (d, 3H,J_(5,6)=6.2 Hz, H-6_(C)); ¹³C NMR β 167.0, 166.0 (2C, CO), 139.1-127.9(Ph), 99.5 (C-1_(E)), 92.3 (C-1_(C)), 81.9 (C-3_(E)), 81.0 (C-2_(E)),79.9 (C-1_(C)), 77.6 (C-4_(E)), 76.0, 75.6, 74.2, 74.1 (4C, OCH₂), 72.1(C-3_(C)), 71.7 (C-4_(E)), 71.1 (C-2_(C)), 68.0 (C-6_(E)), 67.5(C-5_(C)), 41.5 (CH₂Cl), 18.9 (C-6_(C)); FAB-MS for C₄₉H₅₁ClO₁₂ (M,866.3) m/z 889.3 [M+Na]⁺. Anal. Calcd for C₄₉H₅₁ClO₁₂: C, 67.85; H,5.93%. Found: C, 67.72; H, 6.00%.

(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-2-O-benzoyl-3-O-chloroacetyl-α-L-rhamnopyranosyltrichloroacetimidate (119). Trichloroacetonitrile (1.1 mL, 10.9 mmol)and DBU (17 μL) were added to a solution of the hemiacetal 123 (950 mg,1.09 mmol) in dry CH₂Cl₂ (8 mL), and the mixture was stirred at 0° C.for 1.5 h. Toluene was added, and volatiles were evaporated. The residuewas purified by flash chromatography (solvent B, 3:2 containing 0.1%Et₃N) to give 119 (930 mg, 84%) as a colourless foam. Further elutiongave some remaining starting material 123 (136 mg, 14%). [α]_(D)+39.3 (c1.0); ¹H NMR β 8.76 (s, 1H, NH), 8.12-7.17 (m, 25H, Ph), 6.34 (d, 1H,J_(1,2)=1.5 Hz, H-1_(C)), 5.67 (dd, 1H, H-2_(C)), 5.54 (dd, 1H,J_(2,3)=3.4, J_(3,4)=8.8 Hz, H-3_(C)), 4.98 (d, 1H, OCH₂), 4.88 (d, 1H,J_(1,2)=3.4H-1_(E)), 4.84 (d, 1H, J=11.1 Hz, OCH₂), 4.82 (d, 1H, J=11.2Hz, OCH₂), 4.65 (d, 1H, OCH₂), 4.62 (d, 1H, OCH₂), 4.44 (d, 1H, J=11.4Hz, OCH₂), 4.41 (d, 1H, J=11.8 Hz, OCH₂), 4.14 (dq, 1H, J_(4,5)=9.5 Hz,H-5_(C)), 4.11 (d, 1H, J=15.5 Hz, CH₂Cl), 3.98 (d, 1H, CH₂Cl), 3.94 (pt,1H, H-3_(E)), 3.83-3.71 (m, 4H, H-5_(E), 6a_(E), 4_(E), 4_(C)),3.56-3.51 (m, 2H, H-6b_(E), 2_(E)), 1.51 (d, 3H, J_(5,6)=6.2 Hz,H-6_(C)); ¹³C NMR δ 167.1, 165.7, 160.6 (3C, CO), 139.0-127.9 (Ph), 99.9(C-1_(E)), 95.2 (C-1_(C)), 82.1 (C-3_(E)), 80.9 (C-2_(E)), 79.0(C-4_(C)), 77.6 (C-4_(E)), 76.0, 75.6, 74.2, 73.8 (4C, OCH₂), 73.0(C-3_(C)), 71.9 (C-5_(E)), 70.7 (C-5_(C)), 69.2 (C-2_(C)), 68.0(C-6_(E)), 67.7 (C-5_(C)), 41.4 (CH₂Cl), 18.6 (C-5_(C)). Anal. Calcd forC₅₁H₅₁Cl₄NO₁₂: C, 60.54; H, 5.08; N, 1.38%. Found: C, 60.49; H, 5.01; N,1.34%.

Methyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-(2-O-benzoyl-3-O-chloroacetyl-α-L-rhamitopyranosyl)-(1→3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-β-D-glucopyranoside(124). The acceptor (L. A. Mulard, C. Costachel, P. J. Sansonetti, J.Carbohydr. Chem. 2000, 19, 849-877) 118 (500 mg, 1.82 mmol) wasdissolved in CH₂Cl₂ (5.5 mL) and 4 Å-MS (300 mg) were added. The mixturewas cooled to −60° C. and stirred for 15 min. TMSOTf (35 μL, mmol) and asolution of the disaccharide donor 119 (2.39 g, 2.36 mmol) in CH₂Cl₂(7.5 mL) were added. The mixture was stirred for 45 min while thecooling bath was coming back to rt, and for more 3 h at rt. The mixturewas then heated at 65° C. for 1 h 30 min. Et₃N was added and the mixturewas stirred at rt for 20 min, then diluted with CH₂Cl₂ and filteredthrough a pad of Celite. The filtrate was concentrated and purified bycolumn chromatography (solvent B, 85:15→1:1) to give 124 (1.64 g, 80%)as a white powder [α]_(D)+55.1 (c 1.0); ¹H NMR δ 8.06-6.93 (m, 25H, Ph),6.18 (d, 1H, J_(NH,2)=7.3 Hz, NH_(D)), 5.40 (dd, 1H, J_(2,3)=3.5 Hz,H-3_(C)), 5.38 (bs, 1H, H-2_(C)), 4.98 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)),4.94 (bs, 1H, H-1_(C)), 4.94 (d, 1H, OCH₂), 4.93 (d, 1H, J_(1,2)=3.4 Hz,H-1_(E)), 4.83 (d, 2H, J=10.7 Hz, OCH₂), 4.81 (d, 1H, J=10.6 Hz, OCH₂),4.67 (d, 1H, J=11.7 Hz, OCH₂), 4.62 (d, 1H, J=11.4 Hz, OCH₂), 4.47 (m,3H, H-3_(D), OCH₂), 4.22 (dq, 1H, J_(4,5)=9.4, J_(5,6)=6.2 Hz, H-5_(C)),4.10 (d, 1H, J=15.5 Hz, CH₂Cl), 3.96 (m, 2H, H-6a_(D), CH₂Cl), 3.91 (pt,1H, H-3_(E)), 3.82 (m, 2H, H-5_(E), 6b_(D)), 3.72 (m, 3H, H-5a_(E),4_(E), 4_(C)), 3.62 (pt, 1H, J_(3,4) J_(4,5)=9.4 Hz, H-4_(D)), 3.55 (m,2H, H-6b_(E), 2_(E)), 3.51 (s, 3H, OCH₃), 3.41 (m, 1H, H-5_(D)), 3.15(m, 1H, H-2_(D)), 2.04 (s, 3H, C(O)CH₃), 1.51 (s, 3H, C(CH₃)₂), 1.42 (m,6H, H-6_(C), C(CH₃)₂), 1.51 (d, 3H, J_(5,6)=6.2 Hz, H-6_(C)); ¹³C NMR δ171.8, 167.3, 166.1 (3C, CO), 139.0-128.0 (Ph), 101.1 (C-1_(D),J_(CH)<164 Hz), 99.9 (C(CH₃)₂), 99.4 (C-1_(E), J_(CH)>165 Hz), 98.2(C-1_(C), J_(CH)=172 Hz), 81.8 (C-3_(E)), 80.9 (C-2_(E)), 79.0(C-4_(C)*), 77.7 (C-4_(E)*), 76.7 (C-3_(D)), 75.9, 75.3, 74.2, 73.9 (4C,OCH₂), 73.7 (C-4_(D)), 73.4 (C-3_(C)), 71.9 (C-5_(E)), 71.2 (C-2_(C)),68.2 (C-6_(E)), 67.8 (C-5_(C)), 67.4 (C-5_(D)), 62.7 (C-6_(D)), 59.6(C-2_(D)), 57.6 (OCH₃), 41.5 (CH₂Cl), 29.5 (C(CH₃)₂), 27.3 (C(O)CH₃),19.7 (C(CH₃)₂), 18.6 (C-6_(C)); FAB-MS for C₆₁H₇₀ClNO₁₇ (M, 1123.4) m/z1146.5 [M+Na]⁺. Anal. Calcd for C₆₁H₇₀ClNO₁₇: C, 65.15; H, 6.27; N,1.25%. Found: C, 65.13; H, 6.23; N, 1.22%.

Methyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-β-D-glucopyranoside(125). To a solution of the fully protected 124 (1.40 g, 1.25 mmol) in amixture of methanol (18 mL) and pyridine (18 mL) was added thiourea (951mg, 12.5 mmol). The mixture was stirred at 65° C. for 5 h at which timeno TLC (solvent C, 4:1) that no starting material remained. Evaporationof the volatiles and co-evaporation of petroleum ether form the residueresulted in a crude solid which was taken up in a minimum of methanol. Alarge excess of CH₂Cl₂ was added and the mixture was left to stand at 0°C. for 1 h. The precipitate was filtrated on a pad of Celite and thefiltrated was concentrated. Column chromatography of the residue(solvent C, 4:1) gave the trisaccharide acceptor 125 (1.28 g, 97%) as awhite powder. [α]_(D)+33.5 (c 1.0); ¹H NMR δ 8.10-6.96 (m, 25H, Ph),6.09 (d, 1H, J_(NH,2)=7.9 Hz, NH_(D)), 5.26 (dd, 1H, J_(1,2)=1.6,J_(2,3)=3.4 Hz, H-2_(C)), 4.97 (m, 3H, H-1_(C), 1_(E), OCH₂), 4.86 (m,3H, H-1_(D), OCH₂), 4.81 (d, 1H, OCH₂), 4.72 (d, 1H, OCH₂), 4.58 (d, 1H,J=12.2 Hz, OCH₂), 4.51 (d, 1H, J=10.9 Hz, OCH₂), 4.48 (d, 1H, J=12.2 Hz,OCH₂), 4.23 (pt, 1H, J_(2,3)=J_(3,4)=9.4 Hz, H-3_(D)), 4.18-4.10 (m, 2H,H-5_(C), 5_(E)), 4.06-3.95 (m, 3H, H-3_(C), 3_(E), 6a_(D)), 3.80 (pt,1H, J_(5,6b)=J_(6a,6b)=10.4 Hz, H-6b_(D)), 3.66 (m, 2H, H-6a_(E),6b_(E)), 3.62 (dd, 1H, J_(2,3)=9.8, J_(1,2)=4.1 Hz, H-2_(E)), 3.59 (pt,1H, J_(3,4)=J_(4,5)=8.9 Hz, H-4_(E)), 3.55 (pt, 1H, J_(3,4)=J_(4,5)=9.2Hz, H-4_(D)), 3.51 (pt, 1H, J_(3,4)=J_(4,5)=9.3 Hz, H-4_(C)), 3.49 (s,3H, OCH₃), 2.22 (s, 3H, C(O)CH₃), 1.90 (bs, 1H, OH), 1.49 (s, 3H, CMe₂),1.43 (s, 3H, CMe₂), 1.40 (s, 3H, J_(5,6)=6.2 Hz, H-6_(C)); ¹³C NMR δ171.8, 166.6 (2C, CO), 138.9-128.1 (Ph), 101.6 (C-1_(D)), 99.8(C(CH₃)₂), 98.6 (C-1_(E)*), 98.3 (C-1_(C)*), 85.4 (C-4_(C)), 82.0(C-3_(E)), 80.4 (C-2_(E)), 78.2 (C-4_(E)), 77.1 (C-3_(D)), 75.9, 75.5,74.2, 73.9 (4C, OCH₂), 73.6 (C-4_(D)*), 73.5 (C-2_(C)*), 71.7 (C-5_(E)),69.0 (C-6_(E)), 68.3 (C-3_(C)), 67.5 (C-5_(D)), 66.9 (C-5_(C)), 62.7(C-6_(D)), 58.9 (C-2_(D)), 57.5 (OCH₃), 29.5 (C(CH₃)₂), 24.0 (C(O)CH₃),19.7 (C(CH₃)₂), 18.2 (C-6_(C)); FAB-MS for C₅₉H₆₉NO₁₆ (M, 1047.5) m/z1070.4 [M+Na]⁺. Anal. Calcd for C₇₀H₇₆O₁₆: C, 67.61; H, 6.64; N, 1.34%.Found: C, 67.46; H, 6.78; N, 1.24%.

Methyl(3,4-Di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-3-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-β-D-glucopyranoside(129). (a) The trisaccharide acceptor 125 (615 mg, 0.58 mmol) wasdissolved in Et₂O (10 mL) and the solution was cooled to −60° C. TMSOTf(32 μL) and donor 120 (497 mg, 0.88 mmol) in Et₂O (12 mL) were added,and the mixture was stirred for 1 h while the bath was slowly comingback to −20° C. The mixture was stirred for 4 h at this temperature,then at 0° C. overnight. More 120 (50 mg, 88 μmol) was added, and themixture was stirred at rt for 3 h more at 0° C. Et₃N was added, and themixture was concentrated. Column chromatography of the residue (solventB, 9:1→1:1) gave the orthoester 135 (44 mg, 5%) then the fully protected129 (445 mg, 52%) contaminated with the trimethylsilyl side product 126(129/126: 9/1) together with a mixture of 129 and 135 (65 mg, 8%), andthe starting 125 (27 mg, 4%). An analytical sample of compound 129 had[α]_(D)+17.9 (c 1.0); ¹H NMR δ 8.07-7.12 (m, 35H, Ph), 5.96 (d, 1H,J_(NH,2)=7.9 Hz, NH), 5.82 (m, 1H, H-2_(B)), 5.33 (dd, 1H, J_(1,2)=1.8,J_(2,3)=3.2 Hz, H-2_(C)), 5.07 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)), 5.05(d, 1H, J_(1,2)=1.7 Hz, H-1_(B)), 4.98 (d, 1H, OCH₂), 4.97 (bs, 1H,H-1_(C)), 4.91-4.78 (m, 5H, H-1_(D), OCH₂), 4.64 (d, 1H, J=11.6 Hz,OCH₂), 4.60-4.45 (m, 5H, OCH₂), 4.36 (d, 1H, J=11.9 Hz, OCH₂), 4.26 (pt,1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3_(D)), 4.17 (dd, 1H, J_(2,3)=3.4 Hz,H-3_(C)), 4.16 (d, 1H, J=15.1 Hz, CH₂Cl), 4.11 (d, 1H, CH₂Cl), 4.10 (dq,1H, J_(4,5)=9.1, J_(5,6)=6.3 Hz, H-5_(C)), 4.06 (m, 1H, H-5_(E)), 4.00(pt, 1H, J_(3,4)=J_(2,3)=9.4 Hz, H-3_(E)), 3.97 (dd, 1H, J_(5,6a)=5.3,J_(6a,6b)=10.8 Hz, 6a_(D)), 3.89 (m, 1H, H-6a_(E)), 3.88-3.68 (m, 4H,H-6b_(E), 6b_(D), 4_(C), 3_(B)), 3.67 (m, 1H, H-5_(B)), 3.58 (pt, 1H,J_(3,4)=J_(4,5)=9.4 Hz, H-4_(D)), 3.52 (dd, 1H, J_(1,2)=3.3, J_(2,3)=9.8Hz, H-2_(E)), 3.49 (s, 3H, OCH₃), 3.39 (m, 1H, H-5_(D)), 3.30 (m, 2H,H-2_(D), 4_(B)), 2.12 (s, 3H, C(O)CH₃), 1.52 (s, 3H, C(CH₃)₂), 1.42 (s,3H, C(CH₃)₂), 1.33, 0.96 (2d, 3H, J_(5,6)=6.2 Hz, H-6_(B), 6_(C)); ¹³CNMR δ 171.9, 167.0, 166.3 (3C, CO), 138.8-128.0 (Ph), 101.4 (C-1_(D),J_(CH)=164 Hz), 99.9 (C(CH₃)₂), 99.3 (C-1_(C), J_(CH)=168 Hz), 98.3(C-1_(E), J_(CH)=168 Hz), 97.9 (C-1_(B), J_(CH)=171 Hz), 82.1 (C-3_(E)),81.8 (C-2_(E)), 80.4 (bs, C-3_(B)), 80.0 (C-4_(C)), 78.8 (bs, C-4_(E)*),78.3 (C-4_(B)*), 77.7 (C-3_(C)*), 76.9 (C-3_(D)), 75.9, 75.5, 75.3, 74.3(4C, OCH₂), 73.4 (C-4_(D)), 73.2 (OCH₂), 72.7 (C-2_(B)), 72.1 (C-5_(E)),69.1 (C-5_(C)), 67.7 (C-5_(D)*), 67.6 (C-5_(B)*), 62.7 (C-6_(D)), 59.1(C-2_(D)), 57.5 (OCH₃), 41.4 (CH₂Cl), 29.5 (C(CH₃)₂), 24.0 (C(O)CH₃),19.7 (C(CH₃)₂), 18.8, 18.2 (2C, C-6_(B), 6_(C)); FAB-MS for C₈₁H₉₂NClO₂₁(M, 1449.5) m/z 1472.7 [M+Na]⁺. Anal. Calcd for C₈₁H₉₂NClO₂₁: C, 67.05;H, 6.39; N, 0.97%. Found: C, 66.21; H, 6.46; 1.01%.

Compound 135 had [α]_(D)+26.7 (c 0.8); ¹H NMR δ 8.07-7.15 (m, 35H, Ph),5.47 (d, 1H, J_(NH,2)=7.4 Hz, NH_(D)), 5.45 (bs, 1H, H-2_(C)), 5.42 (d,1H, J_(1,2)=2.3 Hz, H-1_(B)), 5.24 (d, 1H, J_(1,2)=3.4 Hz, H-1_(E)),4.94 (d, 1H, J_(1,2)=8.2 Hz, H-1_(D)), 4.91-4.82 (m, 7H, H-1_(C), OCH₂),4.80 (d, 1H, J=11 Hz, OCH₂), 4.75 (d, 1H, J=11.6 Hz, OCH₂), 4.68 (dd,1H, J_(1,2)=2.4, J_(2,3)=4.0 Hz, H-2_(B)), 4.65-4.47 (m, 4H, OCH₂),4.44-4.32 (m, 4H, H-5_(E), 3_(D), 3_(C), OCH₂), 4.15 (m, 1H, H-5_(C)),4.05 (pt, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3_(E)), 4.03 (Pt, 1H,J_(3,4)=J_(4,5)=9.4 Hz, H-4_(C)), 3.94 (dd, 1H, J_(5,6a)=5.3,J_(6a,6b)=10.7 Hz, H-6a_(D)), 3.83-3.75 (m, 4H, H-6a_(E), 6b_(D),CH₂Cl), 3.74-3.70 (m, 3H, H-4_(E), 6_(E), 3_(B)), 3.65 (dd, 1H,J_(1,2)=3.4, J_(2,3)=9.4 Hz, H-2_(E)), 3.48 (Pt, 2H, H-4_(B), 4_(D)),3.46 (s, 3H, OCH₃), 3.38 (m, 1H, H-5_(D)), 3.22 (dq, 1H, J_(4,5)=9.5,J_(5,6)=6.2 Hz, H-5_(B)), 2.88 (m, 1H, H-2_(D)), 1.90 (s, 3H, C(O)CH₃),1.42 (s, 3H, C(CH₃)₂), 1.36 (s, 6H, C(CH₃)₂, H-6_(C)), 1.30 (s, 3H,J_(5,6)=6.3 Hz, H-6_(B)); ¹³C NMR δ 171.8, 166.4 (2C, CO), 139.1-122.5(Ph), 101.0 (C-1_(D), J_(CH)=165 Hz), 99.7 (C(CH₃)₂), 98.3 (C-1_(C),J_(CH)=172 Hz), 97.8 (bs, C-1_(E), J_(CH)=170 Hz), 97.5 (C-1_(B),J_(CH)=176 Hz), 82.2 (C-3_(E)), 80.7 (C-2_(E)), 79.3 (bs, C-4_(B)), 78.8(C-3_(B)), 78.1 (bs, C-4_(E)), 77.3 (C-2_(B)), 76.2 (bs, C-3_(C)), 75.8,75.6, 74.9, 74.6, 73.9 (6C, C-4_(C), OCH₂), 73.5 (2C, C-4_(D), 2_(C)),71.4 (OCH₂), 71.0 (C-3_(D)), 70.7 (2C, C-5_(E), 5_(B)), 69.0 (C-5_(C)),68.8 (C-6_(E)), 67.2 (C-5_(D)), 62.5 (C-6_(D)), 60.0 (C-2_(D)), 57.6(OCH₃), 46.9 (CH₂Cl), 29.5 (C(CH₃)₂), 23.9 (C(O)CH₃), 19.7 (C(CH₃)₂),19.0 (C-6_(B)), 18.4 (C-6_(C)); FAB-MS for C₈₁H₉₂NClO₂₁ (M, 1449.5) m/z1472.7 [M+Na]⁺. Anal. Calcd for C₈₁H₉₂NClO₂₁.H₂O: C, 66.23; H, 6.34; N,0.96%. Found: C, 66.11; H, 6.62; N, 0.85%.

Methyl(2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-β-D-glucopyranoside(130). The trisaccharide acceptor 125 (500 mg, 0.47 mmol) was dissolvedin CH₂Cl₂ (5 mL) and the solution was cooled to −40° C. TMSOTf (21 μL)and donor 105 (328 mg, 0.62 mmol) were added and the mixture was leftunder stirring while the bath was slowly coming back to rt. After 5 h,more 105 (50 mg, 94 μmol) was added and the mixture was stirred at rtfor 1 h more at rt. Et₃N was added and the mixture was concentrated.Column chromatography of the residue (solvent B, 4:1→1:1) gave the fullyprotected 130 (484 mg, 72%) slightly contaminated with the correspondingtrimethylsilyl side-product 126 The 130:126 ratio was estimated to be85:15 from the ¹H NMR spectrum. Eluting next was some residual starting125 (45 mg, 9%), thus based on the consumed acceptor, the estimatedyield of contaminated 130 was 79%. An analytical sample of 130 had[α]_(D)+15.9 (c 0.8); ¹H NMR: δ 8.09-7.14 (m, 35H, Ph), 6.04 (bs, 1H,NH_(D)), 5.76 (m, 1H, H-2_(B)), 5.37 (dd, 1H, J_(1,2)=1.9, J_(2,3)=2.8Hz, H-2_(C)), 5.11 (d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 5.06 (d, 1H,H-1_(B)), 4.96 (bs, 1H, H-1_(C)), 5.02-4.82 (m, 7H, H-1_(D), OCH₂),4.69-4.37 (m, 6H, OCH₂), 4.28 (pt, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3_(D)),4.15 (dd, 1H, J_(2,3)=3.3, J_(3,4)=9.4 Hz, H-3_(C)), 4.13-3.93 (m, 5H,H-5_(E), 6a_(E), 3_(E), 5_(C), 6a_(D)), 3.87-3.76 (m, 5H, H-4_(E),6b_(E), 3_(B), 4_(C), 6b_(D)), 3.68 (dq, 1H, J_(4,5)=9.5 Hz, H-5_(B)),3.57 (pt, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-4_(D)), 3.54 (dd, 1H,J_(2,3)=3.2 Hz, H-2_(E)), 3.48 (s, 3H, OCH₃), 3.40 (m, 1H, H-5_(D)),3.34 (pt, 1H, J_(3,4)=9.7 Hz, H-4_(B)), 3.27 (m, 1H, H-2_(D)), 2.18,2.13 (2s, 6H, C(O)CH₃), 1.51, 1.42 (2s, 6H, C(CH₃)₂), 1.33 (d, 3H,J_(5,6)=6.2 Hz, H-6_(C)), 0.98 (d, 3H, J_(5,6)=6.2 Hz, H-6_(B)); ¹³C NMRδ 171.9, 170.5, 166.3 (3C, CO), 139.3-127.7 (Ph), 101.3 (C-1_(D)), 99.9(C(CH₃)₂), 99.6 (C-1_(B)), 98.4 (C-1_(E)), 98.0 (C-1_(C)), 82.1(C-3_(E)), 81.8 (C-2_(E)), 80.3 (2C, C-3_(C), 4_(B)), 78.7 (bs,C-4_(C)), 78.2 (C-3_(B)*), 77.7 (C-4_(E)*), 76.9 (bs, C-3_(D)), 75.9,75.4, 75.3, 74.3 (4C, OCH₂), 73.4 (C-4_(D)), 73.3 (OCH₂), 72.7(C-2_(C)), 72.1 (C-5_(E)), 70.9 (OCH₂), 69.0 (3C, C-2_(B), 5_(B),6_(E)), 67.8 (C-5_(C)), 67.6 (C-5_(D)), 62.7 (C-6_(D)), 59.2 (C-2_(D)),57.5 (OCH₃), 29.5 (C(CH₃)₂), 24.0, 21.6 (2C, C(O)CH₃), 19.7 (C(CH₃)₂),18.9 (C-6_(C)), 18.2 (C-6_(B)). FAB-MS for C₈₁H₉₃NO₂₁ (M, 1415) m/z1438.6 [M+Na]⁺.

Methyl(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-β-D-glucopyranoside(131). (a) Thiourea (22 mg, 0.29 mmol) was added to the chloroacetylated129 (83 mg, 57 μmol) in MeOH/pyridine (1/1, 2.8 mL), and the mixture washeated overnight at 65° C. Volatiles were evaporated, and the solidresidue thus obtained was taken up in the minimum of MeOH. CH₂Cl₂ wasadded, and the suspension was left standing at 0° C. for 1 h. Theprecipitate was filtered on a pad of Celite, and the filtrate wasconcentrated. Column chromatography of the residue (solvent B, 9:1→1:1)gave the tetrasaccharide acceptor 131 (74 mg, 94%).

(b) The monoacetylated 130 (52 mg, 37 μmol) was dissolved in a mixtureof EtOH (10 mL) and CH₂Cl₂ (100 μL). A freshly prepared 0.4 M ethanolicsolution of guanidine (92 μL, 37 μmol) was added and the mixture wasstirred at rt overnight. Volatiles were evaporated, and the residuetaken up in CH₂Cl₂ was washed with water. The organic phase was driedand concentrated. Column chromatography of the crude product gave 131(42 mg, 83%) as a glassy solid. Compound 131 had [α]_(D)+27.3 (c 1.0);¹H NMR δ 8.24-6.88 (m, 35H, Ph), 5.90 (bs, 1H, NH_(D)), 5.29 (bs, 1H,H-2_(C)), 5.14 (d, 1H, J_(1,2)3.0 Hz, H-1_(E)), 5.06 (d, 1H, J_(1,2)=1.6Hz, H-1_(B)), 5.00-4.95 (m, 3H, H-1_(D), 1_(C), OCH₂), 4.88-4.46 (m, 9H,OCH₂), 4.31 (pt, 1H, J_(2,3)=J_(3,4)=9.4 Hz, H-3_(D)), 4.24 (bs, 1H,H-2_(B)), 4.14-3.08 (m, 3H, H-3_(C), 5_(C), 5_(E)), 4.02 (pt, 1H,J_(2,3)=J_(3,4)=9.3 Hz, H-3_(E)), 3.97 (dd, 1H, J_(5,6a)=5.2,J_(6a,6b)=10.7 Hz, 6a_(D)), 3.80 (m, 2H, H-4_(C), 6b_(D)), 3.71 (m, 2H,H-6a_(E), 6b_(E)), 3.66 (pt, 1H, J_(4,5)=9.5 Hz, H-4_(E)), 3.61-3.55 (m,4H, H-3_(B), 2_(E), 5_(B), 4_(D)), 3.50 (s, 3H, OCH₃), 3.42-3.36 (m, 2H,H-5_(D), 4_(B)), 3.20 (m, 1H, H-2_(D)), 2.85 (bs, 1H, OH), 2.10 (s, 3H,C(O)CH₃), 1.51, 1.41 (2s, 6H, C(CH₃)₂), 1.33 (d, 3H, J_(5,6)=6.2 Hz,H-6_(C)), 1.15 (s, 3H, J_(5,6)=6.2 Hz, H-6_(B)); ¹³C NMR δ 171.7, 166.3(2C, CO), 139.0-127.8 (Ph), 103.1 (C-1_(B)), 101.2 (C-1_(D)), 99.8(C(CH₃)₂), 98.2, 98.1 (2C, C-1_(E), 1_(C)), 82.0 (C-3_(E)), 81.5(C-3_(B)*), 80.6 (C-4_(B)), 79.4 (C-2_(E)*), 79.1 (2C, C-4_(C), 3_(C)),78.2 (C-4_(B)), 76.8 (C-3_(D)), 76.0, 75.5, 74.5, 74.2 (4C, OCH₂), 73.9(C-2_(C)), 73.7 (OCH₂), 73.5 (C-4_(D)), 72.1 (OCH₂), 71.6 (C-5_(E)),69.0 (C-6_(E)), 68.7 (2C, C-2_(B), 5_(B)), 67.9 (C-5_(C)), 67.5(C-5_(D)), 62.7 (C-6_(D)), 59.4 (C-2_(D)), 57.5 (OCH₃), 29.5 (C(CH₃)₂),24.0 (C(O)CH₃), 19.7 (C(CH₃)₂), 19.0 (C-6_(C)), 18.3 (C-6_(B)); FAB-MSfor C₇₉H₉₁NO₂₀ (M, 1373) m/z 1396.5 [M+Na]⁺. Anal. Calcd forC₇₉H₉₁NO₂₀.0.5H₂O: C, 68.56; H, 6.65; N, 1.01%. Found: C, 68.53; H,6.71; N, 1.01%.

Methyl(2-O-Acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-3-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-β-D-glucopyranoside(132). Activated 4 Å molecular sieves and TMSOTf (16 μL) were added to asolution of the tetrasaccharide acceptor 131 (406 mg, 0.29 mmol) in Et₂O(10 mL), and the mixture was stirred at −60° C. for 30 min. The donor105 (234 mg, 0.44 mmol) in CH₂Cl₂ (7 mL) was added, and the mixture wasstirred for 1 h while the bath temperature was reaching rt. After afurther 1 h at this temperature, more 105 (50 mg, 94 μmol) was added,and the mixture was stirred for 1 h before Et₃N was added. Filtrationthrough a pad of Celite and evaporation of the volatiles gave a residuewhich was column chromatographed twice (solvent B, 4:1; then solvent A,17:3) to give 132 (262 mg, 52%) as a white powder; [α]_(D)+25.9 (c 1.0);¹H NMR δ 8.07-7.13 (m, 45H, Ph), 6.03 (bs, 1H, NH_(D)), 5.59 (bs, 1H,H-2_(A)), 5.35 (bs, 1H, H-2_(C)), 5.16 (bs, 1H, H-1_(E)), 5.13 (bs, 1H,H-1_(A)), 5.06 (bs, 1H, H-1_(B)), 5.02-4.97 (m, 4H, H-1_(D), 1_(C),OCH₂), 4.91-4.50 (m, 12H, OCH₂), 4.44-4.32 (m, 4H, H-2_(B), 3_(D),OCH₂), 4.20-3.96 (m, 7H, H-5_(E), 5_(A), 3_(C), 3_(E), 6a_(D), 5_(C),3_(A)), 3.87-3.68 (m, 6H, H-4_(E), 6a_(E), 6b_(E), 6b_(D), 4_(C),3_(B)), 3.64-3.47 (m, 7H, H-5_(B), 4_(D), 2_(E), 4_(A), OCH₃), 3.42 (m,1H, H-5_(D)), 3.34 (pt, 1H, J_(3,4)=J_(4,5)=9.3 Hz, H-4_(B)), 3.17 (m,1H, H-2_(D)), 2.13 (s, 3H, C(O)CH₃), 1.49 (s, 3H, C(CH₃)₂), 1.43 (s, 6H,C(CH₃)₂, H-6_(C)), 1.33 (d, 3H, J_(5,6)=6.1 Hz, H-6_(A)), 1.01 (d, 3H,J_(5,6)=5.8 Hz, H-6_(B)); ¹³C NMR δ 171.9, 170.3, 166.3 (3C, CO),139.2-127.6 (Ph), 101.5 (bs, C-1_(B), J_(CH)=171 Hz), 101.2 (C-1_(D),J_(CH)=163 Hz), 99.8 (C(CH₃)₂), 99.7 (C-1_(A), J_(CH)=171 Hz), 97.9 (2C,C-1_(E), 1_(C), J_(CH)=172, J_(CH)=169 Hz), 82.4 (C-3_(E)), 82.1(C-2_(E)), 80.5 (C-4_(A)), 80.2 (bs, C-3_(C)), 80.1 (C-4_(B)), 79.4,78.1, 78.0 (4C, C-3_(B), 4_(E), 3_(A), 4_(C)), 76.6 (bs, C-3_(D)), 75.9,75.8, 75.4 (3C, OCH₂), 74.8 (2C, C-2_(B), OCH₂), 73.5 (C-4_(D)), 73.4(OCH₂), 73.2 (C-2_(C)), 72.1 (OCH₂), 71.8 (C-5_(A)), 71.2 (OCH₂), 69.4(C-2_(A)), 69.2 (C-5_(B)), 68.9 (C-6_(E)), 68.7 (C-5_(C)), 67.8(C-5_(E)), 67.5 (C-5_(D)), 62.7 (C-6_(D)), 59.6 (bs, C-2_(D)), 57.6(OCH₃), 29.5 (C(CH₃)₂), 24.0, 21.4 (2C, C(O)CH₃), 19.7 (C(CH₃)₂), 19.1(C-6_(A)), 18.8 (C-6_(C)), 18.2 (C-6_(B)); FAB-MS for C₁₀₁H₁₁₅NO₂₅ (M,1741.7) m/z 1765.9 [M+Na]⁺. Anal. Calcd for C₁₀₁H₁₁₅NO₂₅: C, 69.60; H,6.65; N, 0.80%. Found: C, 69.56; H, 6.75; N, 0.73%.

Methylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(102). 50% aq TFA (1 mL) was added at 0° C. to a solution of the fullyprotected pentasaccharide 132 (155 mg, 89 μmol) dissolved in CH₂Cl₂ (4mL). After 1 h at this temperature, volatiles were evaporated. Theresidue (crude 133) was taken up in 0.5M methanolic sodium methoxide (8mL) and the mixture was heated overnight at 55° C. Neutralisation withDowex X8 (H⁺), evaporation of the volatiles, and column chromatographyof the residue gave 134 (121 mg, 98%). Compound 134 (111 mg, 81 μmol)was dissolved in a mixture of ethanol (13 mL) and ethyl acetate (2.6 mL)containing 1N aq HCl (130 μL). Palladium on charcoal (130 mg) was addedand the suspension was stirred under a hydrogen atmosphere for 2 h.Filtration of the catalyst and reverse phase chromatography gave thetarget pentasaccharide (60 mg, 88%) as a slightly yellow foam. RP-HPLCpurification followed by freeze-drying gave pure 102 (36 mg). Compound102 had Rt: 9.63 min (solvent F, 100:0→80:20 over 20 min); [α]_(D)−18.6(c 1.0, methanol); ¹H NMR δ 5.13 (d, 1H, J_(1,2)=3.7 Hz, H-1_(E)), 4.98(bs, 1H, H-1_(B)), 4.90 (d, 1H, J_(1,2)=1.4 Hz, H-1_(A)), 4.72 (d, 1H,J_(1,2)=1.4 Hz, H-1_(C)), 4.39 (d, 1H, J_(1,2)=8.6 Hz, H-1_(D)), 4.09(dq, 1H, J_(4,5)=9.2 Hz, H-5_(C)), 4.00 (m, 2H, H-2_(B), 2_(A)),3.94-3.79 (m, 7H, H-5_(E), 2_(C), 3_(C), 6a_(E), 6a_(D), 2_(D), 3_(A)),3.76-3.65 (m, 7H, H-4_(C), 3_(E), 6b_(E), 6b_(D), 5_(A), 5_(B), 3_(B)),3.52 (pt, 1H, J_(3,4)=8.8 Hz, H-3_(D)), 3.49-3.33 (m, 9H, H-4_(D),2_(E), 4_(A), 4_(B), 5_(D), 4_(E), OCH₃), 1.98 (s, 3H, C(O)CH₃), 1.27(d, 3H, J_(5,6)=6.3 Hz, H-6_(C)), 1.24, 1.23 (d, 3H, H-6_(A), 6_(B));¹³C NMR δ 172.3 (CO), 100.7 (C-1_(A), J_(CH)=171 Hz), 99.6 (2C, C-1_(D),1_(B), J_(CH)=163, J_(CH)=170 Hz), 99.2 (C-1_(C), J_(CH)=170 Hz), 95.7(bs, C-1_(E), J_(CH)=170 Hz), 82.0 (C-3_(D)), 79.1 (C-2_(B)), 79.4 (bs,C-3_(C)), 76.4 (C-5_(D)*), 75.4 (bs, C-4_(C)), 73.0 (C-3_(E)), 72.4 (2C,C-4_(A), 4_(B)), 72.2 (C-5_(E)), 71.7 (C-2_(E)), 71.1 (C-2_(C)), 70.4,70.1, 70.0 (4C, C-2_(A), 3_(A), 3_(B), 4_(E)), 69.7, 69.6, 69.3 (3C,C-5_(A), 5_(B), 5_(C)), 68.8 (C-4_(D)), 61.2, 61.0 (2C, C-6_(D), 6_(E)),57.4 (OCH₃), 55.4 (C-2_(D)), 22.6 (C(O)CH₃), 18.2 (C-6_(C)), 17.2, 17.0(C-6_(A), 6_(B)); HRMS (MALDI) Calcd for C₃₃H₅₇NO₂₃+Na: 858.3219. Found:858.3089.

Methyl(2-O-Acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(136). 50% aq TFA (400 μL) was added to a solution of the fullyprotected tetrasaccharide 130 (57 mg, 40 μmol) in CH₂Cl₂ (1 mL) at 0°C., and the mixture was stirred overnight at this temperature. Volatileswere evaporated and the residue was purified by column chromatography(solvent B, 1:1) to give diol 136 (47 mg, 85%). [α]_(D)+19.5 (c 0.9); ¹HNMR δ 8.10-7.16 (m, 35H, Ph), 5.80 (d, 1H, J=8.8 Hz, NH_(D)), 5.66 (m,1H, H-2_(B)), 5.39 (pt, 1H, J_(1,2)=2.8 Hz, H-2_(C)), 5.01 (m, 2H,H-1_(B), 1_(E)), 4.96 (m, 2H, H-1_(C), OCH₂), 4.90-4.81 (m, 5H, H-1_(D),OCH₂), 4.66-4.41 (m, 7H, OCH₂), 4.18 (dd, 1H, J_(2,3)=2.9, J_(3,4)=7.4Hz, H-3_(C)), 4.10 (pt, 1H, H-3_(D)), 4.08-3.95 (m, 5H, H-5_(E), 3_(E),5_(C)), 3.89-3.64 (m, 8H, H-6a_(D), 6b_(D), 6a_(E), 6b_(E), 3_(B),4_(C), 4_(E), 5_(B)), 3.54-3.49 (m, 5H, H-2_(E), 4_(D), OCH₃), 3.45 (m,1H, H-5_(D)), 3.33 (pt, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-4_(B)), 3.27 (m,1H, H-2_(D)), 2.26 (bs, 1H, OH), 2.17 (s, 6H, C(O)CH₃), 1.99 (bs, 1H,OH), 1.39 (d, 3H, J_(5,6)=6.2 Hz, H-6_(C)), 0.95 (d, 3H, J_(5,6)=6.1 Hz,H-6_(B)); ¹³C NMR δ 171.5, 170.4, 166.1 (3C, CO), 139.1-127.8 (Ph),100.9 (C-1_(D)), 99.7 (2C, C-1_(B)*, 1_(C)), 99.2 (bs, C-1_(E)), 85.0(C-3_(D)), 82.1 (C-3_(E)), 81.3 (bs, C-3_(E)), 80.1 (C-4_(B)), 78.0,77.8 (4C, C-3_(C), 4_(C), 3_(B), 4_(E)), 76.0 (OCH₂), 75.6 (C-5_(D)),75.3, 75.2, 74.4, 73.4 (4C, OCH₂), 72.3 (C-2_(C)), 72.1 (C-5_(C)*), 71.3(C-4_(D)), 71.2 (OCH₂), 69.2 (C-5_(B)), 69.0 (C-5_(E), 2_(B)), 68.4(C-6_(E)), 63.2 (C-6_(D)), 57.4 (2C, C-2_(D), OCH₃), 23.9, 21.0 (2C,C(O)CH₃), 19.1 (C-6_(C)), 18.0 (C-6_(B)). FAB-MS for C₇₈H₈₉NO₂₁ (M,1375.59) m/z 1398.6 [M+Na]⁺. Anal. Calcd for C₇₈H₈₉NO₂₁: C, 68.06; H,6.52; N, 1.02%. Found: C, 68.10; H, 6.62; N, 0.98%.

Methylα-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(103). 1% methanolic sodium methoxide (255 μL) was added to a suspensionof diol 136 (68 mg, 49 μmol) in MeOH (2 mL) and the mixture was heatedovernight at 55° C. TLC (solvent A, 19:1) showed that the startingmaterial had been converted to a more polar product. Neutralisation withDowex X8 (H⁺), evaporation of the volatiles, and column chromatography(solvent A, 24:1) gave tetraol 137 (52 mg, 85%). The latter (48 mg, 39μmol) was dissolved in a mixture of ethanol (5 mL) and ethyl acetate (2mL) containing 1N aq HCl (50 μL). Palladium on charcoal (50 mg) wasadded and the suspension was stirred under a hydrogen atmosphereovernight. TLC (solvent E, 4:1:2) showed the presence of a singleproduct. Filtration of the catalyst and reverse phase chromatography,followed by RP-HPLC purification and freeze-drying gave pure 103 (19 mg,71%). Rt: 9.35 min (solvent F, 100:0→80:20 over 20 min); [α]_(D)+12.5 (c0.8, methanol); ¹H NMR δ 5.09 (d, 1H, J_(1,2)=3.7 Hz, H-1_(E)), 4.89(bs, 1H, H-1_(B)), 4.71 (d, 1H, J_(1,2)=1.1 Hz, H-1_(C)), 4.39 (d, 1H,J_(1,2)=8.6 Hz, H-1_(D)), 4.08 (dq, 1H, J_(4,5)=9.3 Hz, H-5_(C)), 3.96(dd, 1H, J_(1,2)=1.4, J_(2,3)=3.2 Hz, H-2_(B)), 3.88-3.80 (m, 4H,H-2_(C), 3_(C), 6a_(E), 6b_(E), 5_(D)), 3.77-3.62 (m, 6H, H-6a_(D),6b_(D), 3_(B), 5_(B), 2_(D), 4_(C)), 3.59 (pt, 1H, J_(3,4)=J_(4,5)=9.4Hz, H-3_(E)), 3.50 (pt, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-3_(E)), 3.50 (pt,1H, J_(3,4)=J_(4,5)=8.7 Hz, H-3_(D)), 3.47-3.34 (m, 8H, H-2_(E), 4_(E),4_(B), 4_(D), 5_(E), OCH₃), 1.98 (s, 3H, C(O)CH₃), 1.27 (d, 3H,J_(5,6)=6.3 Hz, H-6_(C)), 1.21 (d, 3H, J_(5,6)=6.3 Hz, H-6_(B)); ¹³C NMRδ 174.5 (CO), 103.2 (bs, C-1_(B), J_(CH)=72 Hz), 101.8 (C-1_(D),J_(CH)=160 Hz), 101.5 (C-1_(C), J_(CH)=170 Hz), 98.0 (C-1_(E),J_(CH)=170 Hz), 82.2 (C-3_(D)), 79.1 (bs, C-3_(C)), 76.6 (bs, C-4_(C)),76.4 (C-4_(B)*), 72.9 (C-3_(E)), 72.3, 72.2 (2C, C-4_(D), C-5_(D)),71.87 (C-2_(E)), 71.1 (bs, C-2_(C)), 70.6 (2C, C-2_(B), 3_(B)), 69.7,69.6 (2C, C-5_(E), 5_(B)), 69.2, 68.9 (2C, C-6_(D), 6_(E)), 57.4 (OCH₃),55.4 (C-2_(D)), 22.6 (C(O)CH₃), 18.0 (C-6_(C)), 17.0 (C-6_(B)). HRMS(MALDI) Calcd for C₂₇H₄₇NO₁₉Na: 712.2635. Found: 712.2635.

B—Synthesis of a Pentasaccharide Building Block of the O-SpecificPolysaccharide of Shigella flexneri Serotype 2a: DAB(E)C

Dodecyl3,4,6-tri-O-acétyl-2-deoxy-1-thio-2-trichloroacetamido-β-D-glucopyranoside(205). A mixture of the peracetylated 204 (G. Blatter, J.-M. Beau, J.-C.Jacquinet, Carbohydr. Res. 1994, 260, 189-202) (6.2 g, 12.5 mmol) anddodecanthiol (2.5 mL, 94 mmol), 4 Å molecular sieves and dry 1,2-DCE (90mL) was stirred for 1 h, then cooled to 0° C. BF₃.Et₂O (1.57 mL, 12.5mmol) was added. The stirred mixture was allowed to reach rt in 2 h30.Et₃N was added until neutral pH and the mixture filtered. Afterevaporation, the residue was eluted from a column of silica gel with 2:1cyclohexane-EtOAc to give 205 as a white solid (7.5 g, 93%); [α]_(D)−20°(c 1, CHCl₃). ¹H NMR: δ 6.82 (d, 1H, J_(2,NH)=9.2 Hz, NH), 5.31 (dd, 1H,J_(2,3)=9.9, J_(3,4)=9.6 Hz, H-3), 5.15 (dd, 1H, J_(4,5)=9.6 Hz, H-4),4.68 (d, 1H, J_(1,2)=10.3 Hz, H-1), 4.28 (dd, 1H, J_(5,6a)=5.0,J_(6a,6b)=12.3 Hz, H-6a), 4.17 (dd, 1H, J_(5,6b)=2.3 Hz, H-6b), 4.11(dd, 1H, H-2), 3.75 (m, 1H, H-5), 2.70 (m, 2H, SCH₂), 2.10, 2.05, 2.04(3s, 9H, OAc), 1.65-1.20 (m, 20H, (CH₂)₁₀CH₃), 0.90 (t, 3H, (CH₂)₁₀CH₃).¹³C NMR: δ 171.0, 170.7, 169.3 (C═O), 161.9 (C═OCCl₃), 92.3 (CCl₃), 84.2(C-1), 76.5 (C-5), 73.4 (C-3), 68.6 (C-4), 62.6 (C-6), 55.2 (C-2), 32.3,30.6, 30.0-29.1, 14.5 (S(CH₂)₁₁CH₃), 21.1, 21.0, 20.9 (OAc). FAB-MS forC₂₆H₄₂Cl₃NO₈S (M, 635.0) m/z 658.1 [M+Na]⁺. Anal. Calcd forC₂₆H₄₂Cl₃NO₈S: C, 49.17; H, 6.67; N, 2.21%. Found: C, 49.16; H, 6.71; N,2.13%.

Dodecyl2-deoxy-4,6-O-isopropylidene-1-thio-2-trichloroacetamido-β-D-glucopyranoside(207). A mixture of 205 (5.0 g, 7.87 mmol) in MeOH (15 mL) wasdeacetylated by catalytic MeONa overnight. The solution was neutralizedby IR 120 (H⁺) and filtered. After concentration in vacuo, the residue206 was treated by 2,2-dimethoxypropane (70 mL) and APTS (148 mg, 0.94mmol) in DMF (20 mL). After stirring overnight, the mixture wasneutralized with Et₃N and concentrated. The residue was eluted from acolumn of silica gel with 3:1 cyclohexane-EtOAc to give 207 as a whitesolid (3.45 g, 80%); [α]_(D)−35° (c 1, CHCl₃). ¹H NMR: δ 6.92 (d, 1H,J_(2,NH)=8.0 Hz, NH), 4.77 (d, 1H, J_(1,2)=10.4 Hz, H-1), 3.98 (m, 1H,J_(2,3)=J_(3,4)=9.2 Hz, H-3), 3.88 (dd, 1H, J_(5,6a)=5.4, J_(6a,6b)=10.8Hz, H-6a), 3.70 (dd, 1H, J_(5,6b)=0.5 Hz, H-6b), 3.63 (m, 1H, H-2), 3.53(pt, 1H, J_(4,5)=9.2 Hz, H-4), 3.29 (m, 1H, H-5), 2.98 (s, 1H, OH), 2.60(m, 2H, SCH₂), 1.60-1.10 (m, 20H, (CH₂)₁₀CH₃), 1.45, 1.35 (2s, 6H,C(CH₃)₂), 0.80 (t, 3H, CH₃); ¹³C NMR: δ 162.5 (C═OCCl₃), 100.3(C(CH₃)₂), 92.8 (CCl₃), 84.0 (C-1), 74.6 (C-4), 72.3 (C-3), 71.7 (C-5),62.2 (C-6), 58.3 (C-2), 29.3, 19.5 (C(CH₃)₂), 32.3, 30.8, 30.1-29.5,29.1, 14.5 (SCH₂(CH₂)₁₀CH₃). FAB-MS for C₂₃H₄₀Cl₃NO₅S (M, 548.9) m/z572.2 [M+Na]⁺. Anal. Calcd for C₂₃H₄₀Cl₃NO₅S: C, 50.32; H, 7.34; N,2.55%. Found: C, 50.30; H, 7.40; N, 2.36%.

Dodecyl3-O-acetyl-2-deoxy-4,6-O-isopropylidene-1-thio-2-trichloroacetamido-β-D-glucopyranoside(208). A mixture of 207 (1.07 g, 1.94 mmol) in pyridine (10 mL) wascooled to 0° C. Ac₂O (5 mL) was added and the solution was allowed toreach rt in 2 h. The mixture was then concentrated and pyridine wascoevaporated with toluene. The residue was eluted from a column ofsilica gel with 6:1 cyclohexane-EtOAc with 0.2% of Et₃N to give 208 as awhite solid (1.12 g, 97%): [α]_(D)−62° (c 1, CHCl₃); ¹H NMR: δ 7.51 (d,1H, J_(2,NH)=9.7 Hz, NH), 5.40 (dd, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3),4.62 (d, 1H, J_(1,2)=10.4 Hz, H-1), 4.20 (m, 1H, H-2), 4.01 (dd, 1H,J_(5,6a)=5.2, J_(6a,6b)=10.7 Hz, H-6a), 3.84 (dd, 1H, J_(4,5)=9.7 Hz,H-4), 3.70 (m, 2H, H-5, H-6b), 2.68 (m, 2H, SCH₂), 2.09 (s, 3H, OAc),1.60-1.20 (m, 20H, (CH₂)₁₀CH₃), 1.52, 1.38 (2 s, 6H, C(CH₃)₂), 0.90 (t,3H, SCH₂(CH₂)₁₀CH₃). ¹³C NMR: δ 171.4 (C═O), 161.8 (C═OCCl₃), 99.5(C(CH₃)₂), 92.3 (CCl₃), 84.6 (C-1), 73.6 (C-3), 72.0 (C-4), 71.9 (C-5),62.2 (C-6), 55.0 (C-2), 29.1, 19.3 (C(CH₃)₂), 32.3, 30.7, 30.0-29.0,14.5 (SCH₂(CH₂)₁₀CH₃). FAB-MS for C₂₅H₄₂Cl₃NO₆S (M, 591.0) m/z 614.1[M+Na]⁺. Anal. Calcd for C₂₅H₄₂Cl₃NO₆S: C, 50.80; H, 7.16; N, 2.37%.Found: C, 50.67; H, 7.32; N, 2.24%.

Allyl 3,4-di-O-benzyl-2-O-levulinoyl-α-L-rhamnopyranoside (210). DCC(5.76 g, 28.0 mmol), levulinic acid (2.65 g, 22.8 mmol)) and DMAP (115mg) were added to a solution of alcohol 209 (1.65 g, 4.29 mmol) in THF(70 mL). The suspension was stirred at rt overnight. Et₂O was added andsolids were filtered. The filtrate was concentrated, and the residue waspurified twice from a column of silica gel, eluting first with 99.5:0.5to 98:2 DCM-EtOAc, then with 9:1 cyclohexane-acetone. The target 210(2.00 g 97%)) as a colourless oil slightly contaminated by a less polarproduct. ¹H NMR: δ 7.40-7.30 (m, 10H, Ph), 5.90 (m, 1H, All), 5.40 (dq,1H, J_(1,2)=1.8, J_(2,3)=3.4 Hz, H-2), 5.28 (m, 1H, All), 5.20 (m, 1H,All), 4.93 (d, 1H, J=10.8 Hz, CH₂Ph), 4.78 (d, 1H, J_(1,2)=1.6 Hz, H-1),4.78 (d, 1H, J=11.2 Hz, CH₂Ph), 4.63 (d, 1H, CH₂Ph), 4.51 (d, 1H,CH₂Ph), 4.17 (m, 2H, All, H-3), 3.78 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2Hz, H-5), 3.43 (pt, 1H, J_(3,4)=9.5 Hz, H-4), 2.80 (m, 4H, Lev), 2.19(s, 3H, Ac), 1.37 (d, 3H, H-6). ¹³C NMR: δ 124.0-125.1 (Ph), 118.0(All), 97.0 (C-1), 80.2 (C-4), 78.5 (C-3), 75.2 (CH₂Ph), 72.0 (CH₂Ph),70.2 (C-2), 68.5 (All), 68.3 (C-5), 38.5 (Lev), 31.5 (Ac), 28.5 (Lev),20.1 (C-6). Anal. Calcd for C₂₅H₃₀O₇: C, 69.69; H, 7.10. Found: C,69.61; H, 7.10.

3,4-Di-O-benzyl-2-O-levulinoyl-α-L-rhamnopyranose (211).1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (25 mg, 20 μmol) was dissolved THF and the resultingred solution was degassed in an argon stream. Hydrogen was then bubbledthrough the solution, causing the colour to change to yellow. Thesolution was then degassed again in an argon stream. A solution of 210(1.4 g, 3.12 mmol) in THF was degassed and added. The mixture wasstirred at rt overnight, then concentrated to dryness. The residue wasdissolved in a solution of I₂ (1.37 g, 5.4 mmol) in 30 mL of THF/H₂O(15:4). The mixture was stirred at rt for 1 h, and THF was evaporated.The resulting suspension was taken up in DCM, washed twice with water,satd aq NaHSO₃, water, satd aq NaHCO₃, water and satd aq NaCl,successively. The organic layer was dried and concentrated. The residuewas eluted from a column of silica gel with 7:3 to 6:4 Cyclohexane-EtOActo give the corresponding hemiacetal 211 (1.3 g, 93%). ¹H NMR: δ7.40-7.30 (m, 10H, Ph), 5.40 (dq, 1H, J_(1,2)=1.8, J_(2,3)=3.4 Hz, H-2),4.93 (d, 1H, J=10.8 Hz, CH₂Ph), 4.78 (d, 1H, J_(1,2)=1.6 Hz, H-1), 4.78(d, 1H, J=11.2 Hz, CH₂Ph), 4.63 (d, 1H, CH₂Ph), 4.51 (d, 1H, CH₂Ph),3.99 (m, 1H, J_(3,4)=9.5 Hz, H-3), 3.78 (dq, 1H, J_(4,5)=9.5,J_(5,6)=6.2 Hz, H-5), 3.43 (pt, 1H, H-4), 2.80 (m, 4H, Lev), 2.19 (s,3H, Ac), 1.37 (d, 3H, H-6). Anal. Calcd for C₂₈H₃₄O₇: C, 67.86; H, 6.83.Found: C, 67.94; H, 6.87.

3,4-Di-O-benzyl-2-O-levulinoyl-α-L-rhamnopyranosyl trichloroacetimidate(212). Trichloroacetonitrile (1.3 mL, 13 mmol) and DBU (51 μL, 0.3 mmol)were added to a solution of the residue 211 (1.0 g, 2.3 mmol) inanhydrous DCM (6 mL) at 0° C. After 2 h, the mixture was concentrated.The residue was eluted from a column of silica gel with 3:1cyclohexane-EtOAc and 0.2% Et₃N to give 212 as a white foam (1.0 g,95%); ¹H NMR: δ 8.67 (s, 1H, NH), 7.40-7.30 (m, 10H, Ph), 6.19 (d, 1H,J_(1,2)=1.9 Hz, H-1), 5.48 (dd, 1H, J_(1,2)=2.0, J_(2,3)=3.3 Hz, H-2),4.95 (d, 1H, CH₂Ph), 4.73 (d, 1H, CH₂Ph), 4.66 (d, 1H, CH₂Ph), 4.58 (d,1H, CH₂Ph), 4.51 (d, 1H, CH₂Ph), 4.00 (dd, 1H, J_(3,4)=9.5 Hz, H-3),3.95 (dq, 1H, J_(4,5)=9.6, J_(5,6)=6.3 Hz, H-5), 3.52 (pt, 1H, H-4),2.80 (m, 4H, Lev), 2.20 (s, 3H, Ac), 1.36 (d, 3H, H-6). Anal. Calcd forC₂₇H₃₀Cl₃NO₇.0.5H₂O: C, 54.42; H, 5.24; N, 2.35. Found: C, 54.06; H,5.06; 2.05.

Allyl(2-O-levulinoyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(215). A mixture of alcohol 214 (F. Segat, L. A. Mulard, Tetrahedron:Asymmetry 2002, 13, 2211-2222) (300 mg, 0.36 mmol) and imidate 212 (320mg, 0.54 mmol) in anhydrous Et₂O (20 mL) was stirred for 15 min underdry Ar. After cooling at −75° C., Me₃SiOTf (13 μL, 70 μmol) was addeddropwise and the mixture was stirred 3 h. Et₃N (60 μL) was added and themixture was concentrated. The residue was eluted from a column of silicagel with 9:1 cyclohexane-EtOAc to give 215 (440 mg, 92%) as a colourlessfoam. ¹H NMR: δ 8.10-7.10 (m, 35H, Ph), 5.95 (m, 1H, All), 5.73 (dd, 1H,J_(1,2)=2.2, J_(2,3)=2.3 Hz, H-2_(B)), 5.43 (dd, 1H, J_(1,2)=2.0,J_(2,3)=3.0 Hz, H-2_(C)), 5.30 (m, 2H, All), 5.08 (d, 1H, J_(1,2)=3.2Hz, H-1_(E)), 5.03 (d, 1H, J_(1,2)=1.7 Hz, H-1_(B)), 4.97 (d, 1H,J_(1,2)=1.9 Hz, H-1_(C)), 4.30-5.00 (m, 12H, CH₂Ph), 4.20 (m, 2H, All,H-3_(C)), 4.05 (m, 3H, All, H-3_(E), 5_(E)), 3.98 (m, 1H, H-6a_(E)),3.81 (m, 5H, H-3_(B), 4_(C), 4_(E), 5_(C), 6_(E)), 3.69 (dq, 1H,J_(4,5)=9.3, J_(5,6)=6.0 Hz, H-5_(B)), 3.52 (dd, 1H, J_(2,3)=9.7 Hz,H-2_(E)), 3.29 (dd, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-4_(B)), 2.71 (m, 4H,CH₂CH₂), 2.15 (s, 3H, Ac), 1.40 (d, 3H, H-6_(C)), 1.01 (d, 3H, H-6_(B)).

Allyl(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(216). The trisaccharide 215 (200 mg, 0.16 mmol) was treated with 0.4 mLof a solution 1 M of hydrazine (100 mg) diluted in a mixture of pyridine(1.6 mL) and acetic acid (0.4 mL) at rt. The solution was stirred during20 min. Acetone (1.2 mL) was added and the solution was concentrated.The residue was eluted from a column of silica gel with 98.5:1.5DCM-EtOAc to give 216 (174 mg) as a foam. Although, contaminated withhydrazine salts, the ¹H NMR spectrum showed that compound 216 had NMRdata identical to that of a reference compound. (F. Bélot, K. Wright, C.Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074)

Allyl(2-O-levulinoyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(217). Triflic acid (3.5 μL, 40 μmol) was added to a mixture of thedonor 212 (88 mg, 265 μmol), the acceptor 216 (F. Bélot, K. Wright, C.Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69, 1060-1074)(197 mg, 176 μmol), and 4 Å molecular sieves in dry DCM (2.5 mL) keptunder stirring at −30° C. The suspension was stirred for 1 h at thistemperature, then at rt for 2 h. More 212 (40 mg, 120 mmol) was addedand the mixture was kept at 4° C. for 40 h. After addition of moretriflic acid (1 μL, 11 μmol) and stirring for 2 h at rt, Et₃N was addedto the reaction mixture. Filtration through a pad of Celite, andevaporation of the volatiles resulted in a oily residue which waspurified by flash chromatography with 7:3 cyclohexane-EtOAc to give 217(123 mg, 54%).

¹H NMR: δ 8.10-7.00 (m, 45H, Ph), 5.82 (m, 1H, All), 5.61 (bs, 1H,H-2_(A)), 5.48 (bs, 1H, H-2_(C)), 5.34 (m, 2H, All), 4.97 (bs, 2H,H-1_(B), 1_(E)), 5.10 (bs, 1H, H-1_(C)), 5.02 (bs, 1H, H-1_(A)),5.06-4.37 (m, 16H, CH₂Ph), 4.45 (bs, 1H, H-2_(B)), 4.28-3.83 (m, 8H,H-3_(E), 5_(E), 3_(A), 5_(A), 3_(C), 5_(C)), 3.83 (m, 3H, H-6a_(E),6b_(E), 4_(C)), 3.80 (m, 1H, H-4_(E)), 3.72 (dd, 1H, H-3_(B)), 3.66 (m,1H, H-5_(B)), 3.57 (dd, 1H, H-2_(E)), 3.51 (dd, 1H, H-4_(A)), 3.39 (dd,1H, H-4_(B)), 2.66 (m, 4H, CH₂CH₂), 2.13 (s, 3H, CH₃), 1.45 (2d, 6H,H-6_(A), 6_(C)), 1.07 (d, 3H, H-6_(B)); ¹³C NMR: δ 206.4, 172.1, 166.2(3C, C═O), 139.2-127.6 (Ph), 118.1 (All), 101.4 (C-1_(B)), 99.7(C-1_(A)), 98.3 (C-1_(E)), 96.5 (C-1_(C)), 82.3 (C-3_(E)), 81.5(C-2_(E)), 80.5 (C-3_(C)), 80.2 (2C, C-4_(A), 4_(B)), 79.3 (C-3_(B)),78.6 (C-3_(A)), 78.0 (2C, C-4_(C), 4_(E)), 76.0, 75.8, 75.6 (3C, CH₂Ph),75.2 (C-2_(B)), 75.0, 74.4, 73.4 (3C, CH₂Ph), 72.9 (C-2_(C)), 72.0(CH₂Ph), 71.8 (C-5_(E)), 71.1 (CH₂Ph), 69.8 (C-2_(A)), 69.3 (C-5_(B)),68.9, 68.8 (All, C-6_(E)), 68.7 (C-5_(A)), 68.0 (C-5_(C)), 38.5, 28.5(2C, CH₂CO), 30.2 (CH₃), 19.2, 18.8, 18.2 (3C, C-6_(A), 6_(B), 6_(C)).

Allyl(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(218). The tetrasaccharide 217 (121 mg, 0.09 mmol) was treated with 235μL of a 1 M solution of hydrazine hydrate (100 mg) in a mixture ofpyridine (1.6 mL) and acetic acid (0.4 mL) at rt. The solution wasstirred during 15 min. Acetone (3 mL) was added and the solution wasconcentrated. The residue was eluted from a column of silica gel with9:1 cyclohexane-acetone to give alcohol 218 (70 mg). Compound 218 hadNMR data identical to that of a reference compound. (F. Bélot, K.Wright, C. Costachel, A. Phalipon, L. A. Mulard, J. Org. Chem. 2004, 69,1060-1074)

Allyl(3-O-acetyl-4,6-O-isopropylidene-2-trichloroacetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(219). A mixture of the donor 208 (294 mg, 357 μmol), the acceptor 218(F. Bélot, K. Wright, C. Costachel, A. Phalipon, L. A. Mulard, J. Org.Chem. 2004, 69, 1060-1074) (313 mg, 211 μmol), and 4 Å molecular sievesin dry DCM (4 mL) was stirred for 1.5 h then cooled to −15° C. NIS (94mg, 0.42 mmol) and triflic acid (8 μL, 0.1 mmol) were successivelyadded. The stirred mixture was allowed to reach 0° C. in 1.5 h. Et₃N (25μL) was added and the mixture filtered. After evaporation, the residuewas eluted from a column of silica gel with 6:1 cyclohexane-EtOAc and0.5% of Et₃N to give 219 as a white foam (232 mg, 58%); [α]_(D)−2° (c 1,CHCl₃); ¹H NMR: δ 7.00-8.00 (m, 45H, Ph), 6.81 (d, 1H, J_(2,NH)=9.0 Hz,NH_(D)), 5.82 (m, 1H, All), 5.30 (dd, 1H, J_(1,2)=1.0, J_(2,3)=3.0 Hz,H-2_(C)), 5.10-5.23 (m, 2H, All), 4.96 (bs, 1H, H-1_(A)), 4.91 (d, 1H,J_(1,2)=3.1 Hz, H-1_(E)), 4.87 (d, 1H, J_(1,2)=1.6 Hz, H-1_(B)), 4.84(bs, 1H, H-1_(C)), 4.79 (dd, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3_(D)), 4.35(d, 1H, H-1_(D)), 4.34 (dd, 1H, H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph),4.00 (dd, 1H, H-2_(A)), 3.90 (dd, 1H, H-2_(D)), 2.90-4.10 (m, 22H, All,H-2_(E), 3_(A), 3_(B), 3_(C), 3_(E), 4_(A), 4_(B), 4_(C), 4_(D), 4_(E),5_(A), 5_(B), 5_(C), 5_(D), 5_(E), 6a_(D), 6b_(D), 6a_(E), 6b_(E)), 1.93(s, 3H, OAc), 1.2-0.9 (m, 15H, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³C NMR:δ 170.7, 165.5, 161.7 (C═O), 138.4-117.3 (Ph, All), 101.7 (C-1_(D)),100.8 (C-1_(B)), 100.6 (C-1_(A)), 99.5 (C(CH₃)₂), 97.9 (C-1_(C)), 95.7(C-1_(C)), 92.0 (CCl₃), 82.2, 81.7, 81.6, 80.3, 79.9, 78.8, 77.9, 77.9,76.6, 76.0, 75.8, 75.4, 75.1, 74.7, 74.3, 74.1, 73.3, 72.8, 72.6, 71.9,71.5, 70.8, 69.0, 68.8, 68.5, 68.0, 67.8, 62.0, 56.7 (C-2_(D)), 28.6(C(CH₃)₂), 21.3 (OAc), 19.4 (C(CH₃)₂), 19.0, 18.5, 18.4 (3C, C-6_(A),6_(B), 6_(C)).

Allyl(2-acetamido-3-O-acetyl-4,6-O-isopropylidene-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-]-2-O-benzoyl-α-L-rhamnopyranoside(201). A mixture of 219 (144 mg, 0.06 mmol), Bu₃SnH (0.1 mL, 0.37 mmol)and AlBN (10 mg) in dry toluene (3 mL) was stirred for 1 h at rt under astream of dry Ar, then was heated for 1.5 h at 90° C., cooled andconcentrated. The residue was eluted from a column of silica gel with2:1 cyclohexane-EtOAc and 0.2% of Et₃N to give 201 (100 mg, 74%). ¹HNMR: δ 6.95-8.00 (m, 45H, Ph), 5.82 (m, 1H, All), 5.46 (d, 1H,J_(2,NH)=8.0 Hz, NH_(D)), 5.29 (dd, 1H, J_(1,2)=1.0, J_(2,3)=3.0 Hz,H-2_(C)), 5.11-5.25 (m, 2H, All), 5.00 (bs, 1H, H-1_(A)), 4.90 (d, 1H,J_(1,2)=3.1 Hz, H-1_(E)), 4.85 (d, 1H, J_(1,2)=1.6 Hz, H-1_(B)), 4.83(bs, 1H, H-1_(C)), 4.70 (dd, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3_(D)), 4.44(d, 1H, H-1_(D)), 4.34 (dd, 1H, H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph),4.02 (dd, 1H, H-2_(A)), 3.37 (dd, 1H, H-2_(E)), 2.90-4.10 (m, 21H, All,H-2_(D), 3_(A), 3_(B), 3_(C), 3_(E), 4_(A), 4_(B), 4_(C), 4_(D), 4_(E),5_(A), 5_(B), 5_(C), 5_(D), 5_(E), 6a_(D), 6b_(D), 6a_(E), 6b_(E)), 1.92(s, 3H, OAc), 1.57 (s, 3H, AcNH), 1.27-0.90 (m, 15H, C(CH₃)₂, H-6_(A),6_(B), 6_(C)). ¹³C NMR: δ 171.3, 170.3, 166.2 (C═O), 138.7-117.9 (Ph,All), 103.9 (C-1_(D)), 101.5 (C-1_(B)), 101.4 (C-1_(A)), 99.9 (C(CH₃)₂),98.5 (C-1_(E)), 96.3 (C-1_(C)), 82.1, 81.7, 81.6, 80.3, 80.1, 78.8,78.1, 77.8, 76.0, 75.8, 75.3, 75.1, 74.7, 74.2, 73.6, 73.3, 72.7, 71.9,71.4, 70.8, 69.0, 68.8, 68.7, 68.4, 68.1, 67.8, 62.1, 55.0 (C-2_(D)),30.0 (C(CH₃)₂), 23.5 (AcNH), 21.6 (OAc), 19.2 (C(CH₃)₂), 19.0, 18.3,18.2 (3C, C-6_(A), 6_(B), 6_(C)). FAB-MS for C₁₀₃H₁₁₇NO₂₅ (M, 1769.0)m/z 1791.9 [M+Na]⁺. Anal. Calcd. for C₁₀₃H₁₁₇NO₂₅: C, 69.93; H, 6.67; N,0.79. Found: C, 69.77; H, 6.84; N, 0.72.

(2-Acetamido-3-O-acetyl-4,6-O-isopropylidene-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-]-2-O-benzoyl-α-L-rhamnopyranosyltrichloroacetimidate (203).1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (50 mg, 58 μmol) was dissolved THF (10 mL), and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the colour to change toyellow. The solution was then degassed again in an argon stream. Asolution of 201 (1.8 g, 1.02 mmol) in THF (20 mL) was degassed andadded. The mixture was stirred at rt overnight then concentrated todryness. The residue was dissolved in acetone (9 mL), then water (2 mL),mercuric chloride (236 mg) and mercuric oxide (200 mg) were addedsuccessively. The mixture protected from light was stirred at rt for 2 hand acetone was evaporated. The resulting suspension was taken up inDCM, washed twice with 50% aq KI, water and satd aq NaCl, dried andconcentrated. The residue was eluted from a column of silica gel with3:2 Cyclohexane-EtOAc and 0.2% Et₃N to give the corresponding hemiacetal220. Trichloroacetonitrile (2.4 mL) and DBU (72 μL) were added to asolution of the residue in anhydrous DCM (24 mL) at 0° C. After 1 h, themixture was concentrated. The residue was eluted from a column of silicagel with 3:2 cyclohexane-EtOAc and 0.2% Et₃N to give 203 as a colourlessoil (1.58 g, 82%); [α]_(D)+2° (c 1, CHCl₃). ¹H NMR: δ 8.62 (s, 1H,C═NH), 6.95-8.00 (m, 45H, Ph), 6.24 (d, 1H, J_(1,2)=2.6 Hz, H-1_(C)),5.48 (dd, 1H, J_(2,3)=3.0 Hz, H-2_(C)), 5.41 (d, 1H, J_(2,NH)=8.4 Hz,NH_(D)), 4.99 (bs, 1H, H-1_(A)), 4.92 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)),4.88 (d, 1H, J_(1,2)=1.6 Hz, H-1_(B)), 4.69 (dd, 1H,J_(2,3)=J_(3,4)=10.0 Hz, H-3_(D)), 4.44 (d, 1H, H-1_(D)), 4.34 (dd, 1H,H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph), 4.02 (dd, 1H, H-2_(A)), 3.38 (dd,1H, H-2_(E)), 2.90-4.10 (m, 19H, H-2_(D), 3_(A), 3_(B), 3_(C), 3_(E),4_(A), 4_(B), 4_(C), 4_(D), 4_(E), 5_(A), 5_(B), 5_(C), 5_(D), 5_(E),6a_(D), 6b_(D), 6a_(E), 6b_(E)), 1.95 (s, 3H, OAc), 1.55 (s, 3H, AcNH),1.30-0.85 (m, 15H, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³C NMR: δ 172.4,171.4, 166.9 (C═O), 140.2-128.9 (Ph), 104.2 (C-1_(D)), 101.4 (2C,C-1_(A), 1_(B)), 101.1 (C(CH₃)₂), 98.0 (C-1_(E)), 94.8 (C-1_(C)), 92.4(CCl₃), 82.1, 81.5, 80.2, 80.1, 78.6, 78.1, 77.8, 77.6, 76.0, 75.8,75.5, 75.0, 74.3, 74.2, 73.5 (C-3_(D)), 73.4, 71.9, 71.4, 71.0, 70.5,69.2, 68.8, 68.3, 68.1, 62.1, 54.9 (C-2_(D)), 29.3 (C(CH₃)₂), 23.4(AcNH), 21.4 (OAc), 19.2 (C(CH₃)₂), 19.0, 18.2, 18.1 (3C, C-6_(A),6_(B), 6_(C)). FAB-MS for C₁₀₂H₁₁₃Cl₃N₂O₂₅ (M, 1873.3) m/z 1896.3[M+Na]⁺. Anal. Calcd. for C₁₀₂H₁₁₃Cl₃N₂O₂₅: C, 65.40; H, 6.08; N, 1.50.Found: C, 65.26; H, 6.02; N, 1.31.

C. Convergent Synthesis of the Decasaccharide D′A′B′(E′)C′DAB(E)C as itsMethyl Glycoside

Phenyl(3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-1-thio-α-L-rhamnopyranoside(308). A mixture of alcohol 315 (0.12 g, 0.27 mmol) and imidate 316(0.245 g, 0.41 mmol) in anhydrous DCM (10 mL) was stirred for 15 minunder dry argon. After cooling at 0° C., Me₃SiOTf (28 μL) was addeddropwise and the mixture was stirred for 0.5 h. Triethylamine (60 μL)was added and the mixture was concentrated. The residue was eluted froma column of silica gel with 4:1 cyclohexane-EtOAc to give 308 (227 mg,97%) as a colourless foam; [α]_(D)−63° (c 1, CHCl₃). ¹H NMR: δ 7.40-7.10(m, 15H, Ph), 6.73 (d, 1H, J_(2,NH)=8.5 Hz, NH_(D)), 5.47 (d, 1H,J_(1,2)=1.2 Hz, H-1_(A)), 5.07 (pt, 1H, J_(2,3)=J_(3,4)=10.0 Hz,H-3_(D)), 4.99 (pt, 1H, J_(4,5)=10.0 Hz, H-4_(D)), 4.80-4.55 (m, 4H,CH₂Ph), 4.52 (d, 1H, J_(1,2)=8.2 Hz, H-1_(D)), 4.13-3.95 (m, 2H,J_(5,6)=5.3, J_(6a,6b)=12.2 Hz, H-6a_(D), 6b_(D)), 4.10 (dq, 1H,J_(4,5)=9.5, J_(5,6)=6.1 Hz, H-5_(A)), 4.00 (dd, 1H, J_(2,3)=3.0 Hz,H-2_(A)), 3.99 (m, 1H, H-2_(D)), 3.77 (dd, 1H, J_(3,4)=9.4 Hz, H-3_(A)),3.50 (m, 1H, H-5_(D)), 3.39 (dd, 1H, H-4_(A)), 1.95, 1.93, 1.90 (3s, 9H,OAc), 1.23 (d, 3H, H-6_(A)); ¹³C NMR (CDCl₃) δ 171.1, 170.9, 169.6,162.1 (C═O), 138-127 (Ph), 102.1 (C-1_(D)), 92.7 (CCl₃), 87.4 (C-1_(A)),81.3 (C-4_(A)), 80.5 (C-3_(A)), 79.1 (C-2_(A)), 76.4, 74.1 (2C, CH₂Ph),72.4 (C-5_(D)), 72.4 (C-3_(D)), 69.8 (C-5_(A)), 68.7 (C-4_(D)), 62.3(C-6_(D)), 56.2 (C-2_(D)), 21.0, 20.9, 20.8 (3C, OAc), 18.1 (C-6_(A)).FAB-MS for C₄₀H₄₄Cl₃NO₁₂S (M, 867), m/z 890 [M+Na]⁺. Anal. Calcd forC₄₀H₄₄Cl₃NO₁₂S: C, 55.27; H, 5.10; N, 1.61. Found: C, 55.16; H, 5.18; N,1.68.

Allyl(3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(317). A mixture of alcohol 314 (1.86 g, 4.86 mmol) and imidate 316(3.85 g, 6.47 mmol) in anhydrous CH₃CN (80 mL) was stirred for 15 minunder dry Ar. After cooling at 0° C., Me₃SiOTf (46 μL) was addeddropwise and the mixture was stirred for 0.5 h. Triethylamine (150 μL)was added and the mixture was concentrated. The residue was eluted froma column of silica gel with 7:3 cyclohexane-EtOAc to give 317 (4.0 g,99%) as a white solid; [α]_(D)−3° (c 1, CHCl₃). ¹H NMR: δ 7.32-7.18 (m,10H, Ph), 6.70 (d, 1H, J_(2,NH)=8.4 Hz, NH_(D)), 5.82-5.78 (m, 1H, All),5.20-5.05 (m, 2H, All), 5.00 (m, 2H, H-3_(D), 4_(D)), 4.75-4.45 (m, 4H,CH₂Ph), 4.76 (d, 1H, J_(1,2)=1.1 Hz, H-1_(A)), 4.60 (d, 1H, J_(1,2)=8.5Hz, H-1_(D)), 4.15-4.05 (m, 2H, J_(5,6)=4.8, J_(6a,6b)=12.2 Hz,H-6a_(D), 6b_(D)), 3.98 (m, 1H, H-2_(D)), 3.90 (m, 2H, All), 3.86 (dd,1H, J_(2,3)=3.2 Hz, H-2_(A)), 3.81 (dd, 1H, J_(3,4)=9.5 Hz, H-3_(A)),3.62 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.1 Hz, H-5_(A)), 3.50 (m, 1H,H-5_(D)), 3.32 (pt, 1H, H-4_(A)), 2.02, 1.97, 1.93 (3 s, 9H, OAc), 1.24(d, 3H, H-6_(A)); ¹³C NMR: δ 171.0, 170.9, 169.6, 162.1 (C═O),138.5-117.1 (Ph, All), 101.8 (C-1_(D)), 98.5 (C-1_(A)), 92.6 (CCl₃),81.4 (C-4_(A)), 80.4 (C-3_(A)), 77.1 (C-2_(A)), 75.9, 74.1 (2C, CH₂Ph),72.7 (C-3_(D)), 72.5 (C-5_(D)), 68.6 (C-4_(D)), 68.3 (C-5_(A)), 68.1(All), 62.3 (C-6_(D)), 56.1 (C-2_(D)), 21.1, 20.9, 20.9 (3C, OAc), 18.2(C-6_(A)). FAB-MS for C₃₇H₄₄Cl₃NO₁₃ (M, 815), m/z 838 [M+Na]⁺. Anal.Calcd for C₃₇H₄₄Cl₃NO₁₃: C, 54.39; H, 5.43; N, 1.71%. Found: C, 54.29;H, 5.45; N, 1.72%.

(3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranose(318). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (120 mg, 140 μmol) was dissolved tetrahydrofuran (10mL), and the resulting red solution was degassed in an argon stream.Hydrogen was then bubbled through the solution, causing the colour tochange to yellow. The solution was then degassed again in an argonstream. A solution of 317 (1.46 g, 1.75 mmol) in tetrahydrofuran (20 mL)was degassed and added. The mixture was stirred at rt overnight. Themixture was concentrated. The residue was taken up in acetone (27 mL),and water (3 mL) was added. Mercuric bromide (949 mg, 2.63 mmol) andmercuric oxide (761 mg, 3.5 mmol) were added to the mixture, protectedfrom light. The mixture was stirred for 2 h at rt, then concentrated.The residue was taken up in CH₂Cl₂ and washed three times with sat. aq.KI, then with brine. The organic phase was dried and concentrated. Theresidue was purified by column chromatography (cyclohexane-EtOAc 4:1) togive 318 (1.13 g, 81%) as a white foam. [α]_(D)+4° (c 1, CHCl₃). ¹H NMR:δ 7.35-7.05 (m, 10H, Ph), 6.74 (d, 1H, J_(2,NH)=8.5 Hz, NH_(D)), 5.10(d, 1H, J_(1,2)=1.1 Hz, H-1_(A)), 5.02 (m, 2H, H-3_(D), 4_(D)),4.80-4.50 (m, 4H, CH₂Ph), 4.61 (d, 1H, J_(1,2)=8.5 Hz, H-1_(D)),4.15-4.08 (m, 2H, J_(5,6)=4.5, J_(6a,6b)=12.3 Hz, H-6a_(D), 6b_(D)),4.00 (m, 1H, H-2_(D)), 3.90 (dd, 1H, J_(2,3)=3.3, H-2_(A)), 3.86 (dd,1H, J_(3,4)=9.5 Hz, H-3_(A)), 3.85 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2 Hz,H-5_(A)), 3.50 (m, 1H, H-5_(D)), 3.30 (pt, 1H, H-4_(A)), 2.85 (d, 1H,J_(1,OH)=3.5 Hz, OH), 2.02, 1.97, 1.94 (3s, 9H, OAc), 1.23 (d, 3H,H-6_(A)); ¹³C NMR: δ 171.1, 170.0, 169.6, 162.1 (C═O), 138.5-127.1 (Ph),101.7 (C-1_(D)), 94.1 (C-1_(A)), 92.6 (CCl₃), 81.4 (C-4_(A)), 79.9(C-2_(A)), 77.3 (C-3_(A)), 75.9, 74.1 (2C, CH₂Ph), 72.7 (C-3_(D)), 72.5(C-5_(D)), 68.6 (C-4_(D)), 68.4 (C-5_(A)), 62.2 (C-6_(D)), 56.1(C-2_(D)), 21.1, 21.0, 20.9 (3C, OAc), 18.3 (C-6_(A)). FAB-MS forC₃₄H₄₀Cl₃NO₁₃ (M, 775), m/z 789 [M+Na]⁺. Anal. Calcd for C₃₄H₄₀Cl₃NO₁₃:C, 52.55; H, 5.19; N, 1.80%. Found: C, 52.48; H, 5.37; N, 1.67%.

(3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranosetrichloroacetimidate (306). The hemiacetal 318 (539 mg, 0.68 mmol) wasdissolved in CH₂Cl₂ (50 mL), placed under argon and cooled to 0° C.Trichloroacetonitrile (0.6 mL, 6.8 mmol), then DBU (10 μL, 70 μmol) wereadded. The mixture was stirred at 0° C. for 30 min. The mixture wasconcentrated and toluene was co-evaporated from the residue. The residuewas eluted from a column of silica gel with 7:3 cyclohexane-EtOAc and0.2% of Et₃N to give 306 (498 mg, 78%) as a colourless foam; [α]_(D)−18°(c 1, CHCl₃). ¹H NMR: δ 8.48 (s, 1H, NH), 7.40-7.15 (m, 10H, Ph), 6.75(d, 1H, J_(2,NH)=8.5 Hz, NH_(D)), 6.18 (d, 1H, J_(1,2)=1.1 Hz, H-1_(A)),5.15 (pt, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3_(D)), 5.07 (pt, 1H,J_(4,5)=9.5 Hz, H-4_(D)), 4.82-4.50 (m, 4H, CH₂Ph), 4.62 (d, 1H,J_(1,2)=8.5 Hz, H-1_(D)), 4.20-4.03 (m, 2H, J_(5,6)=4.5, J_(6a,6b)=12.3Hz, H-6a_(D), 6b_(D)), 3.98 (m, 1H, H-2_(D)), 3.85 (dq, 1H, J_(3,4)=9.5,J_(5,6)=6.2 Hz, H-5_(A)), 3.84 (dd, 1H, J_(2,3)=3.3 Hz, H-2_(A)), 3.83(dd, 1H, J_(3,4)=9.5 Hz, H-3_(A)), 3.55 (m, 1H, H-5_(D)), 3.45 (pt, 1H,H-4_(A)), 1.98, 1.96, 1.93 (3s, 9H, OAc), 1.23 (d, 3H, H-6_(A)); ¹³CNMR: δ 171.1, 170.0, 169.6, 162.1 (C═O), 138.4-127.2 (Ph), 101.7(C-1_(D)), 97.2 (C-1_(A)), 92.6 (CCl₃), 80.5 (C-4_(A)), 79.1 (C-3_(A)),76.2 (C-2_(A)), 76.2, 74.1 (2C, CH₂Ph), 74.4 (C-3_(D)), 74.1 (C-5_(D)),71.3 (C-5_(A)), 68.6 (C-4_(D)), 62.3 (C-6_(D)), 56.3 (C-2_(D)), 21.1,21.0, 20.9 (3C, OAc), 18.2 (C-6_(A)). Anal. Calcd for C₃₆H₄₀Cl₆N₂O₁₃: C,46.93; H, 4.38; N, 3.04%. Found: C, 46.93; H, 4.52; N, 2.85%.

Allyl(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(319). A mixture of the protected disaccharide 317 (3.0 g, 3.61 mmol) inMeOH (50 mL) was cold to 0° C. and treated by NH₃ gas overnight. Thesolution was concentrated and the residue (2.02 g) was dissolved againin MeOH (50 mL) and treated by Ac₂O (3.98 mL, 36.1 mol). The solutionwas stirred for 2 h and then concentrated. The residue was eluted from acolumn of silica gel with 95:5 DCM-EtOAC to give the intermediate triolwhich was dissolved in Pyridine (5 mL), cold to 0° C. and treated byAc₂O (2.4 mL). The mixture was stirred overnight and concentrated. Theresidue was eluted from a column of silica gel with 3:2cyclohexane-EtOAc to give 319 (2.3 g, 90%) was obtained as a colourlessfoam. [α]_(D)−12° (c 1, CHCl₃). ¹H NMR: δ 7.32-7.18 (m, 10H, Ph),5.80-5.70 (m, 1H, All), 5.40 (d, 1H, J_(2,NH)=8.1 Hz, NH), 5.20-5.10 (m,2H, All), 4.96 (pt, 1H, J_(3,4)=J_(4,5)=9.5 Hz, H-4_(D)), 4.90 (pt, 1H,J_(2,3)=9.5 Hz, H-3_(D)), 4.80 (d, 1H, J_(1,2)=1.2 Hz, H-1_(A)),4.76-4.52 (m, 4H, CH₂Ph), 4.46 (d, 1H, J_(1,2)=8.5 Hz, H-1_(D)),4.10-4.02 (m, 2H, J_(5,6)=4.7, J_(6a,6b)=11.2 Hz, H-6a_(D), 6b_(D)),3.92 (m, 1H, H-2_(D)), 3.87 (m, 2H, All), 3.86 (dd, 1H, J_(2,3)=3.5 Hz,H-2_(A)), 3.82 (dd, 1H, J_(3,4)=9.5 Hz, H-3_(A)), 3.62 (dq, 1H,J_(4,5)=9.5, J_(5,6)=6.2 Hz, H-5_(A)), 3.52 (m, 1H, H-5_(D)), 3.30 (pt,1H, H-4_(A)), 1.98, 1.94, 1.92 (3 s, 9H, OAc), 1.26 (d, 3H, H-6_(A));¹³C NMR: δ 171.1, 171.0, 170.3, 169.6 (C═O), 138-117 (Ph, All), 103.4(C-1_(D)), 98.5 (C-1_(A)), 81.3 (C-4_(A)), 80.4 (C-3_(A)), 78.5(C-2_(A)), 75.9, 73.9 (2C, CH₂Ph), 73.6 (C-3_(D)), 72.4 (C-5_(D)), 68.7(C-4_(D)), 68.2 (C-5_(A)), 68.1 (All), 62.5 (C-6_(D)), 54.5 (C-2_(D)),23.4 (NHAc), 21.2, 21.1, 21.0 (3C, OAc), 18.1 (C-6_(A)). FAB-MS forC₃₇H₄₇NO₁₃ (M, 713.3) m/z 736.2 [M+Na]⁺. Anal. Calcd for C₃₇H₄₇NO₁₃: C,62.26; H, 6.64; N, 1.96. Found: C, 62.12; H, 6.79; N, 1.87.

(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranose(320). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (10 mg, 12 μmol) was dissolved THF (10 mL), and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the colour to change toyellow. The solution was then degassed again in an argon stream. Asolution of 319 (830 mg, 1.16 mmol) in THF (40 mL) was degassed andadded. The mixture was stirred at rt overnight. The mixture wasconcentrated. The residue was taken up in acetone (90 mL), and water (10mL) was added. Mercuric chloride (475 mg, 1.75 mmol) and mercuric oxide(504 mg, 2.32 mmol) were added to the mixture, protected from light. Themixture was stirred for 2 h at rt, then concentrated. The residue wastaken up in CH₂Cl₂ and washed three times with sat. aq. KI, then withbrine. The organic phase was dried and concentrated. The residue waspurified by column chromatography (cyclohexane-EtOAc 3:7) to give 320(541 mg, 69%) as a white foam; [α]_(D)+16° (c 1.0, CHCl₃); ¹H NMR: δ7.35-7.05 (m, 10H, Ph), 5.50 (d, 1H, J_(2,NH)=8.2 Hz, NH_(D)), 5.22 (d,1H, J_(1,2)=1.1 Hz, H-1_(A)), 5.06 (pt, 1H, J_(3,4)=J_(4,5)=9.5 Hz,H-4_(D)), 5.00 (pt, 1H, J_(2,3)=9.5 Hz, H-3_(D)), 4.85-4.60 (m, 4H,CH₂Ph), 4.56 (d, 1H, J_(1,2)=7.0 Hz, H-1_(D)), 4.22-4.13 (m, 2H,J_(5,6)=4-5, J_(6a,6b)=12.3 Hz, H-6a_(D), 6b_(D)), 4.03 (m, 1H,H-2_(D)), 4.00 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2 Hz, H-5_(A)), 3.96 (dd,1H, J_(2,3)=3.3 Hz, H-2_(A)), 3.90 (dd, 1H, J_(3,4)=9.5 Hz, H-3_(A)),3.60 (m, 1H, H-5_(D)), 3.48 (d, 1H, J_(1,OH)=3.5 Hz, OH), 3.40 (pt, 1H,H-4_(A)), 2.08, 2.03, 2.01 (3s, 9H, OAc), 1.65 (s, 3H, NHAc), 1.30 (d,3H, H-6_(A)); ¹³C NMR: δ 171.2, 171.0, 170.4, 169.6 (C═O), 138.2-128.0(Ph), 103.3 (C-1_(D)), 94.1 (C-1_(A)), 81.4 (C-4_(A)), 79.9 (C-2_(A)),78.7 (C-3_(A)), 75.8, 73.9 (2C, CH₂Ph), 73.6 (C-3_(D)), 72.4 (C-5_(D)),68.7 (C-4_(D)), 68.2 (C-5_(A)), 62.4 (C-6_(D)), 54.5 (C-2_(D)), 23.3(NHAc), 21.1, 21.0, 21.0 (3C, OAc), 18.3 (C-6_(A)). FAB-MS forC₃₄H₄₃NO₁₃ (M, 673.2), m/z 696.3 [M+Na]⁺. Anal Calcd for C₃₄H₄₃NO₁₃: C,60.61; H, 6.43; N, 2.08. Found: C, 60.46; H, 6.61; N, 1.95.

(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranosetrichloroacetimidate (307). The hemiacetal 320 (541 mg, 0.80 mmol) wasdissolved in CH₂Cl₂ (20 mL), placed under argon and cooled to 0° C.Trichloroacetonitrile (0.810 mL, 8 mmol), then DBU (10 μL, 80 μmol) wereadded. The mixture was stirred at 0° C. for 1 h. The mixture wasconcentrated and toluene was co-evaporated from the residue. The residuewas eluted from a column of silica gel with 1:1 cyclohexane-EtOAc and0.2% of Et₃N to give 307 (560 mg, 86%) as a colourless foam; [α]_(D)+20(c 1, CHCl₃). ¹H NMR: δ 8.56 (s, 1H, NH), 7.50-7.20 (m, 10H, Ph), 6.29(d, 1H, J_(1,2)=1.3 Hz, H-1_(A)), 5.50 (d, 1H, J_(2,NH)=8.3 Hz, NH_(D)),5.17 (pt, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3_(D)), 5.09 (dd, 1H,J_(4,5)=9.5 Hz, H-4_(D)), 4.85-4.60 (m, 4H, CH₂Ph), 4.68 (d, 1H,J_(1,2)=8.0 Hz, H-1_(D)), 4.22-4.10 (m, 2H, J_(5,6)=5.0, J_(6a,6b)=12.2Hz, H-6a_(D), 6b_(D)), 4.00 (m, 1H, H-2_(D)), 3.99 (dd, 1H, J_(2,3)=3.5Hz, H-2_(A)), 3.90 (dq, 1H, J_(4,5)=9.6, J_(5,6)=6.2 Hz, H-5_(A)), 3.89(dd, 1H, J_(3,4)=9.5 Hz, H-3_(A)), 3.62 (m, 1H, H-5_(D)), 3.50 (dd, 1H,H-4_(A)), 2.02, 2.00, 1.98 (3s, 9H, OAc), 1.65 (s, 3H, NHAc), 1.32 (d,3H, H-6_(A)); ¹³C NMR: δ 171.2, 171.0, 170.4, 169.6 (C═O), 160.5 (C═NH),138.2-128.0 (Ph), 103.3 (C-1_(D)), 97.3 (C-1_(A)), 91.4 (CCl₃), 80.3(C-4_(A)), 79.9 (C-3_(A)), 77.5 (C-2_(A)), 76.0, 73.8 (2C, CH₂Ph), 73.1(C-3_(D)), 72.2 (C-5_(D)), 71.1 (C-5_(A)), 68.8 (C-4_(D)), 62.5(C-6_(D)), 54.8 (C-2_(D)), 23.3 (NHAc), 21.4, 21.1, 21.0 (3C, OAc), 18.4(C-6_(A)). Anal. Calcd for C₃₆H₄₃Cl₃N₂O₁₃: C, 52.85; H, 5.30; N, 3.42.Found: C, 52.85; H, 5.22; N, 3.47.

Allyl(2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(322). The acceptor 314 (1.78 g, 4.65 mmol) and the trichloroacetimidatedonor 321 (2.96 g, 5.58 mmol) were dissolved in anhydrous ether (100mL). The mixture was placed under argon and cooled to −55° C. TMSOTf(335 μL, 1.86 mmol) was added dropwise. The mixture was stirred at −55°C. to −20° C. over 3 h. Triethylamine (0.75 mL) was added, and themixture was allowed to warm to rt. The mixture was concentrated. Theresidue was purified by column chromatography (cyclohexane:EtOAc, 7:3)to give 322 as a colourless syrup (3.21 g, 92%); [α]_(D)−16° (c 0.55,CHCl₃ lit. Zhang, J.; Mao, J. M.; Chen, H. M.; Cai, M. S. Tetrahedron:Asymmetry 1994, 5, 2283-2290) [α]_(D)−19.3° (c, 1.2, CHCl₃); ¹H NMR: δ7.42-7.30 (m, 20H, Ph), 5.92-5.82 (m, 1H, All), 5.62 (dd, 1H,J_(1,2)=1.6, J_(2,3)=3.2 Hz, H-2_(A)), 5.32-5.20 (m, 2H, All), 5.07 (d,1H, H-1_(A)), 4.82 (d, 1H, J_(1,2)=1.0 Hz, H-1_(B)), 4.95-4.60 (m, 8H,CH₂Ph), 4.20-4.15 (m, 1H, All), 4.09 (d, 1H, J_(2,3)=3.0 Hz, H-2_(B)),4.05 (dd, 1H, J_(3,4)=9.4 Hz, H-3_(A)), 4.05-3.95 (m, 1H, All), 3.96(dd, 1H, J_(3,4)=9.5 Hz, H-3_(B)), 3.89 (dq, 1H, J_(4,5)=9.5,J_(5,6)=6.3 Hz, H-5_(A)), 3.76 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2 Hz,H-5_(B)), 3.52 (m, 1H, H-4_(B)), 3.50 (m, 1H, H-4_(A)), 2.18 (s, 3H,OAc), 1.39 (d, 3H, H-6_(A)), 1.36 (d, 3H, H-6_(B)); ¹³C NMR: δ 170.8(C═O), 138.4-117.1 (Ph, All), 99.5 (C-1_(A)), 98.4 (C-1_(B)), 80.5 (2C,C-4_(A), 4_(B)), 80.0 (C-3_(B)), 78.1 (C-3_(A)), 75.8, 75.7 (2C, CH₂Ph),74.9 (C-2_(B)), 72.5, 72.2 (2C, CH₂Ph), 69.3 (C-2_(A)), 68.6 (C-5_(A)),68.4 (C-5_(B)), 68.0 (All), 21.5 (OAc), 18.4, 18.2 (2C, C-6_(A), 6_(B)).CI-MS for C₄₅H₅₂O₁₀ (M, 752) m/z 770 [M+NH₄]⁺. Anal. Calcd. forC₄₅H₅₂O₁₀: C, 71.79; H, 6.96. Found: C, 70.95; H, 7.01.

Allyl(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(323). A 1M solution of sodium methoxide in methanol (1.1 mL) was addedto a solution of 322 (3.10 g, 4.13 mmol) in methanol. The mixture wasstirred at rt for 3 h. The mixture was neutralised with Amberlite IR-120(H⁺) resin, filtered and concentrated to give 323 (2.72 g, 93%) as acolourless syrup which crystallised on standing; mp 98-99° C.; lit.(Pinto, B. M.; Reimer, K. B.; Morissette, D. G.; Bundle, D. R. J. Org.Chem. 1989, 54, 2650-2656) mp 100° C. (hexane); [α]_(D)−30° (c 0.5,CHCl₃), lit. (Pinto, B. M.; Reimer, K. B.; Morissette, D. G.; Bundle, D.R. J. Org. Chem. 1989, 54, 2650-2656) [α]_(D)−32.5° (c, 0.4, CHCl₃); ¹HNMR: δ 7.42-7.30 (m, 20H, Ph), 5.90-5.80 (m, 1H, All), 5.32-5.20 (m, 2H,All), 5.13 (d, 1H, J_(1,2)=1.4 Hz, H-1_(A)), 4.82 (d, 1H, J_(1,2)=1.6Hz, H-1_(B)), 4.95-4.60 (m, 8H, CH₂Ph), 4.20-4.12 (m, 1H, All), 4.19 (m,1H, J_(2,3)=3.2, J_(2,OH)=1.8 Hz, H-2_(A)), 4.09 (d, 1H, J_(2,3)=3.2 Hz,H-2_(B)), 4.00-3.95 (m, 1H, All), 3.95 (dd, 1H, J_(3,4)=9.4 Hz,H-3_(A)), 3.93 (dd, 1H, J_(3,4)=9.4 Hz, H-3_(B)), 3.87 (dq, 1H,J_(4,5)=9.4, J_(5,6)=6.2 Hz, H-5_(A)), 3.74 (dq, 1H, J_(4,5)=9.4,J_(5,6)=6.2 Hz, H-5_(B)), 3.53 (pt, 1H, H-4_(A)), 3.46 (pt, 1H,H-4_(B)), 2.52 (d, 1H, OH), 1.35 (m, 6H, H-6_(A), 6_(B)); ¹³C NMR: δ138.4-117.1 (Ph, All), 101.2 (C-1_(A)), 98.4 (C-1_(B)), 80.8, 80.4 (2C,C-4_(A), 4_(B)), 80.3 (C-3_(B)), 80.0 (C-3_(A)), 75.8, 75.7 (2C, CH₂Ph),75.0 (C-2_(B)), 72.7, 72.6 (2C, CH₂Ph), 69.1 (C-2_(A)), 68.4 (C-5_(B)),68.3 (C-5_(A)), 68.1 (All), 18.4, 18.3 (2C, C-6_(A), 6_(B)). CI-MS forC₄₃H₅₀O₉ (M, 710) m/z 728 [M+NH₄]⁺.

3,4,6-Tri-O-acetyl-2-deoxy-2-tetrachlorophtalimido-β-D-glucopyranosylTrichloroacetimidate (324) (Castro-Palomino, J. C.; Schmidt, R. R.Tetrahedron Lett. 1995, 36, 5343-5346). Trichloroacetonitrile (2.5 mL)and anhydrous potassium carbonate were added to a suspension of3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophtalimido-α/β-D-glucopyranose(7.88 g, 13.75 mmol) in 1,2-DCE (120 mL). The mixture was stirred at rtovernight. TLC (cyclohexane:EtOAc, 3:2) showed that no starting materialremained. The mixture was filtered through a pad of Celite, and thefiltrate was concentrated to give the target 324 as a slightly brownishsolid (9.08 g, 92%).

Allyl(3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(325). 1,2-DCE (35 mL) was added to the trichloroacetimidate donor 316(2.49 g, 4.20 mmol), the acceptor 323 (2.48 g, 3.50 mmol) and 4 Åpowdered molecular sieves (4 g). The mixture was stirred for 1.5 h at rtunder Argon. The mixture was cooled to −20° C. and TMSOTf (230 μL, 1.26mmol) was added. The temperature was allowed to reach 0° C. over 1 h,and the mixture was stirred for an additional 2 h at this temperature.Triethylamine (0.5 mL) was added and the mixture was allowed to warm tort. The mixture was diluted with DCM and filtered. The filtrate wasconcentrated. The residue was purified by column chromatography with 3:1cyclohexane-EtOAc to give 325 (3.83 g, 96%) as a colourless amorphoussolid: [α]_(D)−6° (c 0.5, CHCl₃); ¹H NMR: δ 7.52-7.28 (m, 20H, Ph), 6.83(d, 1H, J_(2,NH)=8.4 Hz, NH), 5.85 (m, 1H, All), 5.26-5.09 (m, 4H,H-3_(D), 4_(D), All), 4.98 (d, 1H, J_(1,2)=1.4 Hz, H-1_(A)), 4.98-4.58(m, 10H, H-1_(B), 1_(D), CH₂Ph), 4.08 (m, 4H, H-2_(A), 2_(D), 6a_(D),All), 3.91 (m, 5H, H-2_(B), 3_(A), 3_(B), 6b_(D), All), 3.79 (m, 2H,H-5_(A), 5_(B)), 3.45 (m, 3H, H-4_(A), 4_(B), 5_(D)), 2.04, 2.02, 1.97(3s, 9H, OAc), 1.30 (m, 6H, H-6_(A), 6_(B)); ¹³C NMR: δ 170.6, 170.3,169.1, 161.6 (C═O), 138.4-117.1 (Ph, All), 101.3 (C-1_(D)), 100.9(C-1_(A)), 97.6 (C-1_(B)), 92.0 (CCl₃), 80.9, 80.4 (2C, C-4_(A), 4_(B)),79.1, 79.0 (2C, C-3_(A), 3_(B)), 77.3 (C-2_(A)), 76.5 (C-2_(B)), 75.4,75.2, 73.6 (3C, CH₂Ph), 72.2 (C-3_(D)), 71.9 (C-5_(D)), 71.6 (CH₂Ph),68.2 (C-5_(B)*), 67.8 (C-4_(D)), 67.5 (C-5_(A)*), 67.5 (CH₂O), 61.3(C-6_(D)), 55.7 (C-2_(D)), 20.5, 20.4 (3C, OAc), 17.9, 17.7 (2C,C-6_(A), 6_(B)). FAB-MS for C₅₇H₆₆Cl₃NO₁₇ (M, 1141.3) m/z 1164.3[M+Na]⁺. Anal. Calcd. for C₅₇H₆₆Cl₃NO₁₇: C, 59.87; H, 5.82; N, 1.22%.Found: C, 59.87; H, 5.92; N, 1.16%.

Allyl(3,4,6-Tri-O-acetyl-2-deoxy-2-tetrachlorophthalimido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(328). Anhydrous Et₂O (30 mL) and DCM (15 mL) were added to thetrichloroacetimidate donor 324 (3.34 g, 4.66 mmol), the acceptor 323(2.20 g, 3.10 mmol). The mixture was cooled to 0° C. and TMSOTf (85 μL,0.466 mmol) was added dropwise. The mixture was stirred at 0° C. for 1h, then at rt for 3 h. Triethylamine (1 mL) was added and the mixturewas stirred for 10 min, then concentrated. The mixture was taken up inEt₂O and the resulting precipitate was filtered off. The filtrate wasconcentrated. The residue was purified by column chromatography with 7:3cyclohexane-EtOAc to give 328 (2.57 g, 65%) as a colourless amorphoussolid: [α]_(D)+22° (c 1.0, CHCl₃); ¹H NMR (300 MHz): δ 7.42-7.16 (m,20H, Ph), 5.91 (dd, 1H, H-3_(D)), 5.81 (m, 1H, All), 5.24-5.10 (m, 4H,H-1_(D), 4_(D), All), 4.93 (s, 1H, H-1_(A)), 4.81-4.53 (m, 5H, H-1_(B),CH₂Ph), 4.45-4.23 (m, 5H, H-2_(D), CH₂Ph), 4.05 (m, 2H, H-6a_(D), All),3.91-3.58 (m, 8H, H-2_(A), 2_(B), 3_(A), 3_(B), 5_(A), 5_(B), 6b_(D),All), 3.38 (m, 1H, H-5_(D)), 3.21-3.13 (m, 2H, H-4_(A), 4_(B)), 2.05,2.02, 2.00 (3s, 9H, OAc), 1.24 (m, 6H, H-6_(A), 6_(B)); ¹³C NMR (75MHz): δ 170.5, 170.4, 169.3 (C═O), 138.4-117.1 (Ph, All), 101.1(C-1_(A)), 99.9 (C-1_(D)), 97.7 (C-1_(B)), 80.6 (2C, C-4_(A), 4_(B)),79.7, 78.9 (2C, C-3_(A), 3_(B)), 78.2 (C-2_(A)), 76.3 (C-2_(B)), 75.2,75.1, 72.6, 71.3 (4C, CH₂Ph), 71.2 (C-5_(D)), 70.1 (C-3_(D)), 68.4(C-5_(B)*), 68.4 (C-4_(D)), 67.6 (C-5_(A)*), 67.6 (All), 61.3 (C-6_(D)),55.4 (C-2_(D)), 20.6, 20.5 (3C, OAc), 18.0, 17.6 (2C, C-6_(A), 6_(B)).FAB-MS for C₆₃H₆₅Cl₄NO₁₈ (M, 1263.3) m/z 1288.4, 1286.4 [M+Na]⁺. Anal.Calcd. for C₆₃H₆₅Cl₄NO₁₈: C, 59.77; H, 5.17; N, 1.11%. Found: C, 60.19;H, 5.53; N, 1.18%.

Allyl(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-β-L-rhamnopyranoside(326) The trisaccharide 325 (1.71 g, 1.50 mmol) was dissolved in MeOH(20 mL). A 1 M solution of sodium methoxide in methanol (9 mL) andtriethylamine (5 mL) were added, and the mixture was stirred at rt for18 h. The mixture was cooled to 0° C. and acetic anhydride was addeddropwise until the pH reached 6. A further portion of acetic anhydride(0.4 mL) was added, and the mixture was stirred at rt for 30 min. Themixture was concentrated, and toluene was co-evaporated from theresidue. The residue was purified by column chromatography with 95:5DCM-MeOH to give 326 (623 mg, 45%) as a colourless amorphous solid:[α]_(D)−16° (c 0.5, CHCl₃); ¹H NMR (300 MHz): δ 7.48-7.24 (m, 20H, Ph),6.79 (d, 1H, NH), 5.73 (m, 1H, All), 5.12 (m, 3H, H-1_(A), All),4.86-4.52 (m, 9H, H-1_(B), CH₂Ph), 4.34 (d, 1H, H-1_(D)), 4.08-3.79 (m,6H, H-2_(A), 2_(B), 3_(A), 3_(B), All), 3.74-3.53 (m, 3H, H-5_(A),5_(B), 6a_(D)), 3.45-3.24 (m, 6H, H-2_(D), 3_(D), 4_(A), 4_(B), 4_(D),6b_(D)), 3.20 (m, 1H, H-5_(D)), 1.46 (s, 3H, NHAc), 1.24 (m, 6H,H-6_(A), 6_(B)); ¹³C NMR (75 MHz): δ 173.6 (C═O), 137.4-117.3 (Ph, All),103.2 (C-1_(D)), 100.3 (C-1_(A)), 97.9 (C-1_(B)), 81.3, 80.4 (2C,C-4_(A), 4_(B)), 79.9 (2C, C-3_(A), 3_(B)), 79.9 (C-2_(B)*), 78.9(C-3_(D)), 75.7 (C-5_(D)), 75.6, 75.3, 74.5 (3C, CH₂Ph), 73.6(C-2_(A)*), 72.5 (CH₂Ph), 71.9 (C-4_(D)), 68.2, 68.0 (2C, C-5_(A),5_(B)), 67.7 (CH₂O), 62.5 (C-6_(D)), 58.8 (C-2_(D)), 22.3 (NHAc), 18.0,17.8 (2C, C-6_(A), 6_(B)). FAB-MS for C₅₁H₆₃NO₁₄ (M, 913.4) m/z 936.6[M+Na]⁺. Anal. Calcd. for C₅₁H₆₃NO₁₄.H₂O: C, 65.72; H, 7.03; N, 1.50%.Found: C, 65.34; H, 7.03; N, 1.55%.

Allyl(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(327). (a) Pyridine (5 mL) was added to 326 (502 mg, 0.55 mmol) and themixture was cooled to 0° C. Acetic anhydride (3 mL) was added. Themixture was stirred at rt for 18 h. The mixture was concentrated andtoluene was co-evaporated from the residue. The residue was taken up inDCM and washed successively with 5% aq HCl and saturated aq NaHCO₃. Theorganic phase was dried and concentrated to give 327 (538 mg, 94%) as acolourless foam.

(b) THF (3 mL) and ethanol (3.3 mL) were added to 328 (384 mg, 0.30mmol). Ethylenediamine (90 μL, 1.36 mmol) was added and the mixture washeated at 55° C. for 4 h. The mixture was allowed to cool to rt. Aceticanhydride (1.0 mL) was added, and the mixture was stirred at rt for 1.5h. The mixture was concentrated. The residue was taken up in pyridine (5mL) and the mixture was cooled to 0° C. Acetic anhydride (2.5 mL) wasadded. The mixture was stirred at rt for 18 h. The mixture wasconcentrated and toluene was co-evaporated from the residue. The residuewas taken up in DCM, which caused the formation of a white precipitate.The mixture was filtered through a plug of silica gel, eluting with 7:3cyclohexane-acetone. The filtrate was concentrated to give 327 (215 mg,68%) as a colourless foam: [α]_(D)−7° (c 0.5, CHCl₃); ¹H NMR (300 MHz):δ 7.48-7.24 (m, 20H, Ph), 5.84 (m, 1H, All), 5.53 (d, 1H, NH), 5.19 (m,2H, All), 5.03 (dd, 1H, H-4_(D)), 4.98 (m, 2H, H-1_(A), 3_(D)),4.95-4.54 (m, 10H, H-1_(B), 1_(D), CH₂Ph), 4.07 (m, 4H, H-2_(A), 2_(D),6a_(D), All), 3.88 (m, 5H, H-2_(B), 3_(A), 3_(B), 6b_(D), All), 3.79,3.68 (2m, 2H, H-5_(A), 5_(B)), 3.42 (m, 3H, H-4_(A), 4_(B), 5_(D)),2.02, 2.01, 1.97, 1.64 (4s, 12H, OAc, NHAc), 1.30 (m, 6H, H-6_(A),6_(B)); ¹³C NMR (75 MHz): δ 170.7, 170.4, 169.9, 169.1 (C═O),138.5-117.1 (Ph, All), 102.9 (C-1_(D)), 101.2 (C-1_(A)), 97.7 (C-1_(B)),81.0, 80.5 (2C, C-4_(A), 4_(B)), 79.5, 79.1 (2C, C-3_(A), 3_(B)), 78.2(C-2_(A)), 76.1 (C-2_(B)), 75.5, 75.2, 73.6 (CH₂Ph), 73.3 (C-3_(D)),71.9 (C-5_(D)), 71.7 (CH₂Ph), 68.3 (C-5_(A)*), 68.0 (C-4_(D)), 67.6(C-5_(B)*), 67.6 (CH₂O), 61.6 (C-6_(D)), 54.1 (C-2_(D)), 22.9 (NHAc),20.7, 20.6 (3C, OAc), 18.0, 17.7 (2C, C-6_(A), 6_(B)). FAB-MS forC₅₇H₆₉NO₁₇ (M, 1039.5) m/z 1062.4 [M+Na]⁺. Anal. Calcd. for C₅₇H₆₉NO₁₇:C, 65.82; H, 6.69; N, 1.35%. Found: C, 65.29; H, 6.82; N, 1.29%.

(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α/β-L-rhamnopyranose(329). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (30 mg, 35 μmol) was dissolved THF (5 mL), and theresulting red solution was processed as described for the preparation of318. A solution of 327 (805 mg, 0.775 mmol) in THF (10 mL) was degassedand added. The mixture was stirred at rt overnight, then concentrated.The residue was taken up in acetone (15 mL) and water (1.5 mL). Mercuricchloride (315 mg, 1.16 mmol) and mercuric oxide (335 mg, 1.55 mmol) wereadded. The mixture, protected from light, was stirred for 1 h at rt,then concentrated. The residue was taken up in DCM and washed threetimes with satd aqueous KI, then with brine. The organic phase was driedand concentrated. The residue was purified by column chromatography with2:3 EtOAc-cyclohexane to give 329 (645 mg, 83%) as a white foam. The ¹HNMR spectra showed the α:β ratio to be 3.3:1; [α]_(D)+3° (c 0.5, CHCl₃);¹H NMR (300 MHz) α-anomer: δ 7.47-7.30 (m, 20H, Ph), 5.53 (d, 1H, NH),5.17 (d, 1H, J_(1,2)=1.9 Hz, H-1_(B)), 5.08 (m, 1H, H-4_(D)), 5.03 (d,1H, J_(1,2)=1.5 Hz, H-1_(A)), 4.99 (m, 1H, H-3_(D)), 4.92-4.62 (m, 8H,CH₂Ph), 4.60 (d, 1H, J_(1,2)=8.4 Hz, H-1_(D)), 4.18-4.01 (m, 3H,H-2_(A), 2_(D), 6a_(D)), 3.97-3.90 (m, 5H, H-2_(B), 3_(A), 3_(B),5_(A)*, 6b_(D)), 3.83 (m, 1H, H-5_(B)*), 3.45-3.37 (m, 3H, H-4_(A),4_(B), 5_(D)), 2.04, 2.03, 1.99, 1.68 (4s, 12H, OAc, NHAc), 1.32 (m, 6H,H-6_(A), 6_(B)); ¹³C NMR (75 MHz) α-anomer: δ 170.7, 170.4, 169.9, 169.1(C═O), 138.5-129.3 (Ph), 103.3 (C-1_(D)), 101.6 (C-1_(A)), 93.9(C-1_(B)), 81.5, 80.8 (2C, C-4_(A), 4_(B)), 79.9, 78.9 (2C, C-3_(A),3_(B)), 78.6 (C-2_(A)), 76.8 (C-2_(B)), 76.0, 75.5, 74.0 (3C, CH₂Ph),73.7 (C-3_(D)), 72.4 (C-5_(D)), 72.2 (CH₂Ph), 68.7 (C-5_(A)*), 68.5(C-4_(D)), 68.2 (C-5_(B)*), 62.0 (C-6_(D)), 54.6 (C-2_(D)), 23.4 (NHAc),21.1, 21.0 (3C, OAc), 18.5, 18.1 (2C, C-6_(A), 6_(B)). FAB-MS forC₅₄H₆₅NO₁₇ (M, 999.4) m/z 1022.5 [M+Na]⁺. Anal. Calcd. for C₅₄H₆₅NO₁₇:C, 64.85; H, 6.55; N, 1.40%. Found: C, 64.55; H, 7.16; N, 1.15%.

(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α/β-L-rhamnopyranosylTrichloroacetimidate (313). The hemiacetal 329 (595 mg, 0.59 mmol) wasdissolved in DCM (10 mL), placed under Argon and cooled to 0° C.Trichloroacetonitrile (0.6 mL, 6 mmol), then DBU (10 μL, 59 μmol) wereadded. The mixture was stirred at 0° C. for 20 min, then at rt for 20min. The mixture was concentrated and toluene was co-evaporated from theresidue. The residue was purified by flash chromatography with 1:1cyclohexane-EtOAc and 0.2% of Et₃N to give 313 (634 mg, 94%) as acolorless foam. The ¹H NMR spectra showed the α:β ratio to be 10:1:[α]_(D)−20° (c 1, CHCl₃); ¹H NMR (300 MHz) α-anomer: δ 8.47 (s, 1H,C═NH), 7.38-7.20 (m, 20H, Ph), 6.10 (d, 1H, J_(1,2)=1.3 Hz, H-1_(B)),5.40 (d, 1H, NH), 5.01 (m, 1H, H-4_(D)), 4.95 (d, 1H, J_(1,2)=1.2 Hz,H-1_(A)), 4.89 (m, 1H, H-3_(D)), 4.85-4.55 (m, 9H, H-1_(D), CH₂Ph), 4.07(dd, 1H, H-6a_(D)), 4.03 (m, 1H, H-2_(A)), 3.97 (m, 1H, H-2_(D)), 3.91(dd, 1H, H-6b_(D)), 3.85-3.71 (m, 5H, H-2_(B), 3_(A), 3_(B), 5_(A),5_(B)), 3.45-3.31 (m, 3H, H-4_(A), 4_(B), 5_(D)), 1.99, 1.96, 1.91, 1.58(4s, 12H, OAc, NHAc), 1.26 (m, 6H, H-6_(A), 6_(B)); ¹³C NMR (75 MHz): δ171.1, 170.9, 170.3, 169.6 (C═O), 160.6 (C═NH), 138.6-128.1 (Ph), 103.3(C-1_(D)), 101.6 (C-1_(A)), 96.9 (C-1_(B)), 91.3 (CCl₃), 81.4, 80.2 (2C,C-4_(A), 4_(B)), 79.9, 78.5 (2C, C-3_(A), 3_(B)), 78.3 (C-2_(A)), 75.9(2C, CH₂Ph), 75.0 (C-2_(B)), 73.7 (CH₂Ph), 73.7 (C-3_(D)), 72.4 (CH₂Ph),72.4 (C-5_(D)), 71.0, 69.0 (2C, C-5_(A), 5_(B)), 68.5 (C-4_(D)), 62.1(C-6_(D)), 54.6 (C-2_(D)), 23.4 (NHAc), 21.1, 21.0 (3C, OAc), 18.5, 18.0(2C, C-6_(A), 6_(B)). Anal. Calcd. for C₅₆H₆₅Cl₃N₂O₁₇: C, 58.77; H,5.72; N, 2.45%. Found: C, 58.78; H, 5.83; N, 2.45%.

Allyl(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(305). Anhydrous Et₂O (5 mL) was added to the donor 313 (500 mg, 0.44mmol) and the acceptor 311 (Segat, F.; Mulard, L. A. Tetrahedron:Asymmetry 2002, 13, 2211-2222) (242 mg, 0.29 mmol) and powdered 4 Åmolecular sieves. The mixture was placed under Argon and cooled to 0° C.Boron trifluoride etherate (415 μL, 3.27 mmol) was added. The mixturewas stirred at 0° C. for 1 h, then at rt for 18 h. The mixture wasdiluted with DCM and triethylamine (1 mL) was added. The mixture wasfiltered through a pad of Celite and the filtrate was concentrated. Theresidue was purified by column chromatography with 3:2 cyclohexane-EtOActo give, in order, the acceptor 311 (132 mg, 54%), 305 (231 mg, 44%) andthe hemiacetal 329 (129 mg, 29%). The desired pentasaccharide 305 wasobtained as a colourless foam: [α]_(D)+10° (c 1.0, CHCl₃); ¹H NMR: δ8.02-7.09 (m, 45H, Ph), 5.92 (m, 1H, All), 5.65 (d, 1H, NH), 5.37 (m,1H, H-2_(C)), 5.19 (m, 2H, All), 5.13 (bs, 1H, H-1_(A)), 4.96-4.35 (m,15H, H-1_(B), 1_(C), 1_(D), 1_(E), 2_(B), 3_(D), 4_(D), CH₂Ph), 4.17 (m,2H, H-2_(A), All), 4.04-3.87 (m, 8H, H-2_(D), 3_(A), 3_(C), 3_(E),5_(A), 5_(E), 6a_(D), All), 3.81-3.63 (m, 7H, H-3_(B), 4_(C), 4_(E),5_(C), 6a_(E), 6b_(E), 6b_(D)), 3.59 (m, 1H, H-5_(B)), 3.43 (m, 3H,H-2_(E), 4_(A), 5_(D)), 3.28 (pt, 1H, H-4_(B)), 2.01, 1.99, 1.71, 1.66(4s, 12H, OAc, NHAc), 1.34 (m, 6H, H-6_(A), 6_(C)), 1.00 (d, 3H,H-6_(B)); ¹³C NMR: δ 170.5, 170.0, 169.3, 165.8, 163.5 (C═O),138.7-117.6 (Ph, All), 102.7 (C-1_(D)), 100.8 (2C, C-1_(A), 1_(B)), 98.1(C-1_(E)), 95.9 (C-1_(C)), 81.8 (C-3_(E)), 81.2 (2C, C-2_(E), 4_(A)),80.0 (C-4_(B)), 79.7 (2C, C-3_(A), 3_(C)), 78.2 (C-3_(B)), 77.7(C-2_(A)), 77.3 (2C, C-4_(C), 4_(E)), 75.6, 75.4, 74.9 (CH₂Ph), 74.3(C-2_(B)), 73.8 (CH₂Ph), 73.7 (C-3_(D)), 72.8 (CH₂Ph), 72.3 (C-2_(C)),72.1 (C-5_(D)), 71.5 (C-5_(E)), 70.2 (CH₂Ph), 68.5 (C-5_(B)), 68.4(C-5_(A), CH₂O), 68.2 (C-4_(D)), 67.9 (C-6_(E)), 67.4 (C-5_(C)), 61.8(C-6_(D)), 54.3 (C-2_(D)), 23.1 (NHAc), 20.7, 20.6, 20.4 (3C, OAc), 18.6(C-6_(A)), 18.0 (C-6_(C)), 17.8 (C-6_(B)). FAB-MS for C₁₀₄H₁₁₇NO₂₇ (M,1812.1) m/z 1836.2, 1835.2 [M+Na]⁺. Anal. Calcd. for C₁₀₄H₁₁₇NO₂₇: C,68.90; H, 6.50; N, 0.77%. Found: C, 68.64; H, 6.66; N, 1.05%.

Allyl(3,4-Di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(332). To a mixture of 323 (3.8 g, 5.35 mmol) in pyridine (40 mL) wasadded chloroacetic anhydride (1.83 g, 10.7 mmol) at 0° C. The solutionwas stirred overnight at 0° C. MeOH (10 mL) was added and the mixturewas concentrated. The residue was eluted from a column of silica gelwith 95:5 cyclohexane-acetone to give 332 (2.4 g, 57%) as a colorlesssyrup: [α]_(D)−15° (c 1.0, CHCl₃); ¹H NMR: δ 7.30-7.15 (m, 20H, Ph),5.81-5.71 (m, 1H, All), 5.49 (dd, 1H, J_(1,2)=1.7, J_(2,3)=3.2 Hz,H-2_(A)), 5.20-5.08 (m, 2H, All), 4.90 (d, 1H, H-1_(A)), 4.84-4.50 (m,8H, PhCH₂), 4.65 (d, 1H, J_(1,2)<1.0 Hz, H-1_(B)), 4.04-3.85 (m, 2H,All), 4.02 (m, 2H, CH₂Cl), 3.93 (dd, 1H, J_(2,3)=3.0 Hz, H-2_(B)), 3.88(dd, 1H, J_(3,4)=9.5 Hz, H-3_(A)), 3.81 (pt, 1H, J_(3,4)=9.5 Hz,H-3_(B)), 3.73 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2 Hz, H-5_(A)), 3.62 (dq,1H, J_(4,5)=9.0, J_(5,6)=6.1 Hz, H-5_(B)), 3.34 (dd, 1H, H-4_(B)), 3.30(dd, 1H, H-4_(A)), 1.22 (d, 3H, H-6_(A)), 1.21 (d, 3H, H-6_(B)); ¹³CNMR: δ 166.9 (C═O), 138.5-117.2 (Ph, All), 99.2 (C-1_(A)), 98.2(C-1_(B)), 80.4 (C-4_(A)), 80.3 (C-3_(B)), 80.2 (C-4_(B)), 77.9(C-3_(A)), 75.8, 75.7, 72.6, 72.4 (4C, PhCH₂), 74.9 (C-2_(B)), 71.2(C-2_(A)), 68.6 (C-5_(A)), 68.4 (C-5_(B)), 68.0 (All), 41.3 (CH₂Cl),18.3 (2C, C-6_(A), 6_(B)). FAB-MS for C₄₅H₅₁ClO₁₀ (M, 786.3) m/z 809.3[M+Na]⁺. Anal. Calcd for C₄₅H₅₁ClO₁₀: C, 68.65; H, 6.53%. Found: C,68.51; H, 6.67%.

(3,4-Di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α/β-L-rhamnopyranose(333). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (40 mg, 46 μmol) was dissolved THF (7 mL), and theresulting red solution was processed as described for the preparation of318. A solution of 332 (2.39 g, 3.04 mmol) in THF (18 mL) was degassedand added. The mixture was stirred at rt overnight. The mixture wasconcentrated. The residue was taken up in acetone (30 mL) and water (5mL). Mercuric chloride (1.24 g, 4.56 mmol) and mercuric oxide (1.3 g,6.08 mmol) were added. The mixture, protected from light, was stirredfor 2 h at rt, then concentrated. The residue was taken up in DCM andwashed three times with satd aqueous KI, then with brine. The organicphase was dried and concentrated. The residue was purified by columnchromatography (cyclohexane-EtOAc, 4:1) to give 333 (1.91 g, 84%) as awhite foam: [α]_(D)−2° (c 1.0, CHCl₃); ¹H NMR: δ 7.40-7.10 (m, 20H, Ph),5.49 (dd, 1H, J_(1,2)=1.7, J_(2,3)=3.2 Hz, H-2_(A)), 4.99 (d, 1H,J_(1,2)<1.0 Hz, H-1_(B)), 4.90 (d, 1H, H-1_(A)), 4.85-4.45 (m, 8H,PhCH₂), 4.01 (m, 2H, CH₂Cl), 3.93 (dd, 1H, J_(2,3)=3.0 Hz, H-2_(B)),3.90 (dd, 1H, J_(3,4)=9.3 Hz, H-3_(A)), 3.84 (dd, 1H, J_(3,4)=9.0 Hz,H-3_(B)), 3.81 (dq, 1H, J_(4,5)=9.0 Hz, J_(5,6)=6.2 Hz, H-5_(B)), 3.72(dq, 1H, J_(4,5)=9.5, J_(5,6)=6.2 Hz, H-5_(A)), 3.33 (pt, 1H, H-4_(B)),3.30 (dd, 1H, H-4_(A)), 2.81 (d, 1H, J_(2,OH)=3.4 Hz, OH), 1.22 (d, 3H,H-6_(A)), 1.20 (d, 3H, H-6_(B)); ¹³C NMR: δ 167.0 (C═O), 138.5-127.2(Ph), 99.1 (C-1_(A)), 93.9 (C-1_(B)), 80.3 (C-4_(B)), 80.2 (C-4_(A)),79.7 (C-3_(B)), 77.8 (C-3_(A)), 75.8, 75.7, 72.6, 72.4 (4C, PhCH₂), 75.0(C-2_(B)), 71.1 (C-2_(A)), 68.6 (C-5_(A)), 68.4 (C-5_(B)), 41.3 (CH₂Cl),18.1 (2C, C-6_(A), 6_(B)). FAB-MS for C₄₂H₄₇ClO₁₀ (M, 746.3) m/z 769.3[M+Na]⁺. Anal. Calcd for C₄₂H₄₇ClO₁₀: C, 67.51; H, 6.34%. Found: C,67.46; H, 6.39%.

(3,4-Di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranosylTrichloroacetimidate (334). The hemiacetal 333 (1.80 g, 2.41 mmol) wasdissolved in DCM (25 mL), placed under Argon and cooled to 0° C.Trichloroacetonitrile (2.4 mL, 24 mmol), then DBU (35 mL, 0.24 mmol)were added. The mixture was stirred at 0° C. for 40 min. The mixture wasconcentrated and toluene was co-evaporated from the residue. The residuewas eluted from a column of silica gel with 4:1 cyclohexane-EtOAc and0.2% Et₃N to give 334 (1.78 g, 83%) as a colorless foam: [α]_(D)−12° (c1.0, CHCl₃); ¹H NMR: δ 8.60 (s, 1H, NH), 7.50-7.30 (m, 20H, Ph), 6.21(d, 1H, J_(1,2)=1.8 Hz, H-1_(B)), 5.63 (dd, 1H, J_(1,2)=1.5, J_(2,3)=3.2Hz, H-2_(A)), 5.07 (d, 1H, H-1_(A)), 5.00-4.65 (m, 8H, PhCH₂), 4.19 (m,2H, CH₂Cl), 4.09 (dd, 1H, J_(2,3)=3.2 Hz, H-2_(B)), 4.04 (dd, 1H,J_(3,4)=9.0 Hz, H1-3_(B)), 3.95 (m, 3H, H-3_(A), 5_(A), 5_(B)), 3.58(dd, 1H, H-4_(A)), 3.48 (dd, 1H, H-4_(B)), 1.39 (m, 6H, H-6_(A), 6_(B));¹³C NMR: δ 167.1 (C═O), 160.7 (C—N), 138.3-127.0 (Ph), 99.4 (C-1_(A)),97.5 (C-1_(B)), 91.4 (CCl₃), 80.1 (C-4_(B)), 80.0 (C-4_(A)), 79.2(C-3_(A)), 77.9 (C-3_(B)), 75.9, 75.8, 73.0, 72.6 (4C, PhCH₂), 73.7(C-2_(B)), 71.4 (C-2_(A)), 71.2, 68.9 (2C, C-5_(A), 5_(B)), 41.3(CH₂Cl), 18.4, 18.2 (2C, C-6_(A), 6_(B)). Anal. Calcd for C₄₄H₄₇Cl₄NO₁₀:C, 59.27; H, 5.31; N, 1.57%. Found: C, 59.09; H, 5.49; N, 1.53%.

Allyl(3,4-Di-O-benzyl-2-O-pmethoxybenzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(335). The alcohol 323 (3.8 g, 5.35 mmol) was dissolved in DMF (25 mL).The mixture was cold to 0° C. and NaH (320 mg, 8.02 mmol) was added in 3parts each 10 min. Then pMeOBnCl (1.8 mL, 13.34 mmol) was added and themixture was stirred overnight at rt. MeOH (5 mL) was added and thesolution stirred for 10 min. The solution was concentrated and theresidue was eluted from a column of silica gel with 95:5cyclohexane-acetone to give 335 (4.34 g, 97%) as a colorless syrup:[α]_(D)−8° (c 1.0, CHCl₃); ¹H NMR (300 MHz): δ 7.20-6.80 (m, 24H, Ph),5.90-5.80 (m, 1H, All), 5.30-5.15 (m, 2H, All), 5.12 (d, 1H, J_(1,2)<1.0Hz, H-1_(A)), 4.73 (d, 1H, J_(1,2)<1.0 Hz, H-1_(B)), 4.70-4.40 (m, 10H,PhCH₂), 4.20-4.08 (m, 1H, All), 4.10 (dd, 1H, J_(2,3)=3.0 Hz, H-2_(B)),3.95-3.88 (m, 3H, H-3_(A), 3_(B), All), 3.80-3.78 (m, 2H, J_(4,5)=9.4,J_(5,6)=6.1 Hz, H-2_(A), 5_(A)), 3.72 (s, 3H, OCH₃), 3.70 (m, 1H,J_(4,5)=9.4, J_(5,6)=6.1 Hz, H-5_(B)), 3.61 (dd, 1H, H-4_(A)), 3.32 (dd,1H, H-4_(B)), 1.18 (d, 3H, H-6_(A)), 1.10 (d, 3H, H-6_(B)); ¹³C NMR (75MHz): δ 133.9-113.8 (Ph, All), 99.0 (C-1_(A)), 97.8 (C-1_(B)), 80.4(C-4_(A)), 80.2 (C-4_(B)), 80.0 (C-3_(B)), 79.0 (C-3_(A)), 75.2, 72.3,71.8, 71.5, 71.3, 67.5 (5C, PhCH₂, All), 74.1 (C-2_(A)), 73.8 (C-2_(B)),68.3 (C-5_(A)), 67.8 (C-5_(B)), 55.0 (OCH₃), 17.8, 17.9 (2C, C-6_(A),6_(B)). FAB-MS for C₅₁H₅₈O₁₀ (M, 830.4) m/z 853.5 [M+Na]⁺. Anal. Calcd.for C₅₁H₅₈O₁₀: C, 73.71; H, 7.03%. Found: C, 73.57; H, 7.21%.

(3,4-Di-O-benzyl-2-O-pmethoxybenzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranose(336). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (50 mg, 60 μmol) was dissolved THF (6 mL), and theresulting red solution was processed as described for the preparation of318. A solution of 335 (4.23 g, 5.09 mmol) in THF (24 mL) was degassedand added. The mixture was stirred at rt overnight, then concentrated.The residue was taken up in acetone (45 mL), and water (5 mL) was added.Mercuric chloride (2.07 g, 7.63 mmol) and mercuric oxide (2.2 g, 10.2mmol) were added. The mixture, protected from light, was stirred for 2 hat rt, then concentrated. The residue was taken up in DCM and washedthree times with satd aqueous KI, then with brine. The organic phase wasdried and concentrated. The residue was purified by columnchromatography (cyclohexane-EtOAc, 4:1) to give 336 (2.97 g, 73%) as awhite foam: [α]_(D)+8° (c 1.0, CHCl₃); ¹H NMR (300 MHz): δ 7.40-7.25 (m,20H, Ph), 7.18-6.73 (m, 4H, Ph), 5.12 (d, 1H, J_(1,2)<1.0 Hz, H-1_(A)),5.05 (d, 1H, J_(1,2)<1.0 Hz, H-1_(B)), 4.80-4.40 (m, 10H, PhCH₂), 4.08(dd, 1H, J_(2,3)=3.0 Hz, H-2_(B)), 3.90-3.80 (m, 2H,J_(3,4)=J_(4,5)=9.5, J_(5,6)=6.1 Hz, H-3_(B), 5_(B)), 3.80-3.78 (m, 2H,J_(2,3)=3.1, J_(4,5)=9.4, J_(5,6)=6.1 Hz, H-2_(A), 5_(A)), 3.73 (m, 1H,J_(3,4)=9.4 Hz, H-3_(A)), 3.72 (s, 3H, OCH₃), 3.60 (pt, 1H, H-4_(A)),3.33 (pt, 1H, H-4_(B)), 1.34 (d, 3H, H-6_(A)), 1.24 (d, 3H, H-6_(B));¹³C NMR (75 MHz): δ 113.2-129.8 (Ph), 99.1 (C-1_(A)), 93.8 (C-1_(B)),80.7 (C-4_(A)), 80.3 (C-4_(B)), 79.7 (C-3_(B)), 79.2 (C-3_(A)), 75.5,75.4, 72.6, 72.5, 72.4 (5C, PhCH₂), 74.2 (C-2_(A)), 74.1 (C-2_(B)), 68.5(C-5_(A)), 68.1 (C-5_(B)), 55.3 (OCH₃), 18.1 (2C, C-6_(A), 6_(B)).FAB-MS for C₄₈H₅₄O₁₀ (M, 790.4) m/z 813.4 [M+Na]⁺. Anal. Calcd. forC₄₈H₅₄O₁₀: C, 72.89; H, 6.88%. Found: C, 72.86; H, 6.98%.

(3,4-Di-O-benzyl-2-O-pmethoxybenzyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α/β-L-rhamnopyranosylTrichloroacetimidate (337). The hemiacetal 336 (2.1 g, 2.66 mmol) wasdissolved in DCM (20 mL), placed under Argon and cooled to 0° C.Trichloroacetonitrile (2.7 mL, 26 mmol), then DBU (40 μL, 0.26 mmol)were added. The mixture was stirred at 0° C. for 30 min. The mixture wasconcentrated and toluene was co-evaporated from the residue. The residuewas eluted from a column of silica gel with 8:2 cyclohexane-EtOAC and0.2% Et₃N to give 337 (2.03 g, 82%) as a colorless foam: [α]_(D)−10° (c1.0, CHCl₃); ¹H NMR (300 MHz): δ 8.50 (s, 1H, NH), 7.25-7.05 (m, 20H,Ph), 7.05-6.62 (m, 4H, Ph), 6.08 (d, 1H, J_(1,2)<1.0 Hz, H-1_(B)), 5.10(d, 1H, J_(1,2)<1.0 Hz, H-1_(A)), 4.80-4.40 (m, 10H, PhCH₂), 4.10 (dd,1H, J_(2,3)=3.0 Hz, H-2_(B)), 3.90-3.80 (m, 4H, H-3_(B), 2_(A), 3_(A),5_(A)), 3.80-3.72 (m, 1H, H-5_(B)), 3.72 (s, 3H, OCH₃), 3.63 (pt, 1H,J_(3,4)=J_(4,5)=9.5 Hz, H-4_(A)), 3.42 (pt, 1H, J_(3,4)=J_(4,5)=9.5 Hz,H-4_(B)), 1.30 (d, 3H, H-6_(B)), 1.25 (d, 3H, H-6_(A)). ¹³C NMR (75MHz): δ 161.1 (C═NH), 129.5-113.4 (Ph), 99.6 (C-1_(A)), 97.0 (C-1_(B)),80.6 (C-4_(A)), 79.6 (C-4_(B)), 79.3 (2C, C-3_(A), 3_(B)), 75.7, 75.5,72.8, 72.3, 72.0 (5C, PhCH₂), 74.4 (C-2_(A)), 72.6 (C-2_(B)), 71.1(C-5_(A)), 68.9 (C-5_(B)), 55.3 (OCH₃), 18.1 (2C, C-6_(A), 6_(B)). Anal.Calcd. for C₅₀H₅₄Cl₃NO₁₀: C, 64.21; H, 5.82; N, 1.50%. Found: C, 64.67;H, 6.01; N, 1.28%.

Allyl(3,4-Di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(338). A mixture of alcohol 311 (212 mg, 0.255 mmol) and imidate 334(270 mg, 0.33 mmol) in anhydrous Et₂O (4 mL) was stirred for 15 minunder dry Argon. After cooling at −60° C., TMSOTf (30 μL, 0.166 mmol)was added dropwise and the mixture was stirred overnight and allowed toreach rt. Triethylamine (120 μL) was added and the mixture wasconcentrated. The residue was eluted from a column of silica gel with7:1 cyclohexane-EtOAc to give 338 (86 mg, 22%) as a foam: [α]_(D)+5° (c1.0, CHCl₃); ¹H NMR (300 MHz) δ 8.00-6.95 (m, 45H, Ph), 6.00-5.80 (m,1H, All), 5.56 (dd, 1H, H-2_(A)), 5.40 (dd, 1H, J_(1,2)<1.0, J_(2,3)=3.0Hz, H-2_(C)), 5.37-5.20 (m, 2H, All), 5.08 (d, 1H, J_(1,2)=3.2 Hz,H-1_(E)), 5.04 (d, 1H, J_(1,2)<1.0 Hz, H-1_(A)), 5.00 (d, 1H,J_(1,2)<1.0 Hz, H-1_(B)), 4.99 (d, 1H, H-1_(C)), 4.90-4.30 (m, 16H,CH₂Ph), 4.35 (dd, 1H, J_(2,3)=3.0 Hz, H-2_(B)), 4.14 (dd, 1H,J_(3,4)=9.5 Hz, H-3_(C)), 4.03 (pt, 1H, J_(2,3)=J_(3,4)=10.0 Hz,H-3_(E)), 4.20-3.90 (m, 2H, All), 4.00-3.75 (m, 4H, CH₂Cl, H-6a_(E),6b_(E)), 3.96 (dd, 1H, H-3_(A)), 3.95 (m, 1H, H-5_(A)), 3.95 (m, 1H,H-5_(E)), 3.83 (dd, 1H, H-4_(C)), 3.80 (m, 1H, H-5_(C)), 3.72 (dd, 1H,H-4_(E)), 3.64 (dd, 1H, H-3_(B)), 3.60 (m, 1H, H-5_(B)), 3.52 (dd, 1H,H-2_(E)), 3.39 (dd, 1H, H-4_(A)), 3.30 (dd, 1H, H-4_(B)), 1.35 (d, 1H,H-6_(A)), 1.30 (d, 1H, H-6_(C)), 1.00 (d, 1H, H-6_(B)); ¹³C NMR (75 MHz)δ 166.1, 165.7 (C═O), 133.4-117.0 (Ph), 100.9 (C-1_(B)), 98.9 (C-1_(A)),97.8 (C-1_(E)), 96.0 (C-1_(C)), 81.8 (C-3_(E)), 80.9 (C-2_(E)), 79.9(C-4_(A)), 79.6 (C-4_(B)), 79.6 (C-3_(C)), 78.9 (C-3_(B)), 78.0(C-4_(C)), 77.5 (C-4_(E)), 77.3 (C-3_(A)), 75.6, 75.3, 75.0, 74.7, 73.9,73.5, 72.8, 70.9 (9C, CH₂Ph, All), 74.9 (C-2_(B)), 72.5 (C-2_(C)), 71.2(C-5_(E)), 70.9 (C-2_(A)), 68.8 (C-5_(B)), 68.5 (C-6_(E)), 68.3(C-5_(A)), 67.5 (C-5_(C)), 40.9 (CH₂Cl), 18.8 (C-6_(A)), 18.2 (C-6_(C)),17.8 (C-6_(B)). FAB-MS for C₉₂H₉₉ClO₂₀ (M, 1558.6) m/z 1581.7 [M+Na]⁺.Anal. Calcd. for C₉₂H₉₉ClO₂₀: C, 70.82; H, 6.40%. Found: C, 70.67; H,6.58%.

Allyl(3,4-Di-O-benzyl-2-O-pmethoxybenzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(339). A mixture of alcohol 311 (125 mg, 0.15 mmol) and 4 Å molecularsieves in anhydrous Et₂O (3 mL) was stirred for 45 min under dry Argon.After cooling at −40° C., Me₃SiOTf (20 μL, 0.112 mmol) was addeddropwise. A solution of the donor 337 (210 mg, 0.225 mmol) in anhydrousEt₂O (2 mL) was added dropwise to the solution of the acceptor during 1h. The mixture was stirred for 3 h at −40° C. Triethylamine (100 μL) wasadded and the mixture was filtered and concentrated. The residue waseluted from a column of silica gel with 85:15 cyclohexane-EtOAc to give339 (107 mg, 44%) as a foam: [α]_(D)+12° (c 1.0, CHCl₃); ¹H NMR: δ8.10-7.10 (m, 45H, Ph), 7.00-6.50 (m, 4H, CH₂PhOMe), 5.90-5.70 (m, 1H,All), 5.32 (dd, 1H, J_(1,2)=1.6, J_(2,3)=3.1 Hz, H-2_(C)), 5.25-5.10 (m,2H, All), 5.05 (d, 1H, H-1_(B)), 4.98 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)),4.85 (m, 2H, H-1_(A), 1_(C)), 4.80-4.20 (m, 18H, CH₂Ph), 4.20-3.90 (m,2H, All), 4.20-3.00 (m, 20H, H-2_(A), 2_(B), 2_(E), 3_(A), 3_(B), 3_(C),3_(E), 4_(A), 4_(B), 4_(C), 4_(E), 5_(A), 5_(B), 5_(C), 5_(E), 6a_(E),6b_(E), OCH₃), 1.30-0.82 (3 d, 9H, H-6_(A), 6_(B), 6_(C)); ¹³C NMR: δ166.3 (C═O), 138.5-118.2 (Ph, All), 99.5, 99.3 (2C, C-1_(A), 1_(B)),98.4 (C-1_(E)), 96.4 (C-1_(C)), 82.3, 81.4, 81.1, 80.5, 80.3, 79.5,78.2, 77.6 (8C, C-2_(E), 3_(A), 3_(B), 3_(C), 3_(E), 4_(A), 4_(B),4_(C)), 76.0, 75.5, 75.3, 74.9, 74.3, 73.3, 72.3, 71.8, 71.6 (9C,CH₂Ph), 74.1, 73.8 (2C, C-2_(A), 2_(B)), 72.5 (C-2_(C)), 72.0 (C-4_(E)),69.2, 69.0, 68.9 (3C, C-5_(A), 5_(B), 5_(C)), 68.8, 68.6 (All, C-6_(E)),67.8 (C-5_(E)), 55.5 (OCH₃), 19.0, 18.8, 18.4 (3C, C-6_(A), 6_(B),6_(C)). FAB-MS for C₉₈H₁₀₆O₂₀ (M, 1603.8) m/z 1626.6 [M+Na]⁺.

Allyl(3,4-Di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(310). A solution of the trisaccharide 342 (Segat, F.; Mulard, L. A.Tetrahedron: Asymmetry 2002, 13, 2211-2222) (8.0 g, 6.5 mmol) in MeOH(128 mL) was treated with 5.7 mL of HBF₄/Et₂O at rt. The solution wasstirred during 4 days. Et₃N was added until neutralization andconcentrated. The residue was diluted with DCM, washed with satd aqNaHCO₃ and water. The organic layer was dried on MgSO₄, filtered andconcentrated. The residue was eluted from a column of silica gel with15:1 toluene-EtOAc to give 310 (6.31 g, 84%) as a foam: [α]_(D)+14° (c1.0, CHCl₃); ¹H NMR: δ 8.10-7.05 (m, 35H, Ph), 5.82 (m, 1H, All), 5.25(dd, 1H, J_(1,2)=1.7, J_(2,3)=3.1 Hz, H-2_(C)), 5.19 (m, 2H, All), 5.00(d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.87 (d, 1H, J_(1,2)=1.8 Hz, H-1_(B)),4.81 (d, 1H, H-1_(C)), 4.90-4.35 (m, 12H, CH₂Ph), 4.20-4.00 (m, 2H,All), 4.10 (dd, 1H, J_(3,4)=8.5 Hz, H-3_(C)), 4.09 (dd, 1H, J_(2,3)=3.2Hz, H-2_(B)), 3.95 (m, 1H, J_(4,5)=9.5 Hz, H-5_(E)), 3.92 (pt, 1H,J_(2,3)=9.5=J_(3,4)=9.5 Hz, H-3_(E)), 3.78 (dq, 1H, J_(5,6)=6.0 Hz,H-5_(C)), 3.70 (m, 1H, H-4_(C)), 3.62-3.58 (m, 2H, H-6a_(E), 6b_(E)),3.59 (m, 1H, J_(4,5)=9.0, J_(5,6)=6.2 Hz, H-5_(B)), 3.54 (dd, 1H,H-4_(E)), 3.48 (dd, 1H, J_(3,4)=8.5 Hz, H-3_(B)), 3.45 (dd, 1H,H-2_(E)), 3.31 (dd, 1H, H-4_(B)), 2.68 (d, 1H, J_(2,OH)=2.3 Hz, OH),1.29 (d, 3H, H-6_(C)), 1.09 (d, 3H, H-6_(B)); ¹³C NMR: δ 166.2 (C═O),137.5-118.2 (Ph, All), 103.1 (C-1_(B)), 98.5 (C-1_(E)), 96.6 (C-1_(C)),82.1 (C-3_(E)), 81.4 (C-2_(E)), 80.4 (C-4_(B)), 79.7 (C-3_(B)), 79.4(C-4_(C)), 78.9 (C-3_(C)), 78.1 (C-4_(E)), 76.0, 75.5, 74.5, 74.2, 73.6,72.1 (6C, CH₂Ph), 73.7 (C-2_(C)), 71.6 (C-2_(B)), 68.9 (C-6_(E)), 68.8(C-5_(B)), 68.7 (All, C-5_(E)), 68.1 (C-5_(C)), 19.1 (C-6_(C)), 18.2(C-6_(B)). FAB-MS for C₇₀H₇₆O₁₅ (M, 1156.5) m/z 1179.5 [M+Na]⁺. Anal.Calcd for C₇₀H₇₆O₁₅: C, 72.64; H, 6.62%. Found: C, 72.49; H, 6.80%.

Allyl(2-O-Acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(344). A mixture of alcohol 310 (5.2 g, 4.49 mmol), imidate 321 (3.58 g,6.74 mmol) and 4 Å molecular sieves in anhydrous Et₂O (117 mL) wasstirred for 1 h under dry Argon. After cooling at −30° C., Me₃SiOTf (580μL, 3.2 mmol) was added dropwise and the mixture was stirred and allowedto rt overnight. Triethylamine (1.2 mL) was added and the mixture wasfiltered and concentrated. The residue was eluted from a column ofsilica gel with 9:1 cyclohexane-EtOAc to give 344 (6.16 g, 90%) as awhite foam: [α]_(D)+130 (c 1.0, CHCl₃); ¹H NMR: δ 8.10-7.00 (m, 45H,Ph), 5.82 (m, 1H, All), 5.45 (dd, 1H, J_(1,2)=1.5, J_(2,3)=2.5 Hz,H-2_(A)), 5.29 (dd, 1H, J_(1,2)=1.5, J_(2,3)=2.5 Hz, H-2_(C)), 5.19 (m,2H, All), 4.97 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)), 4.95 (d, 1H, H-1_(A)),4.91 (d, 1H, J_(1,2)=1.6 Hz, H-1_(B)), 4.84 (d, 1H, H-1_(C)), 4.90-4.35(m, 16H, CH₂Ph), 4.29 (dd, 1H, J_(2,3)=2.6 Hz, H-2_(B)), 4.10-4.00 (m,2H, All), 4.02 (dd, 1H, J_(3,4)=8.5 Hz, H-3_(C)), 3.90 (m, 2H,J_(2,3)=J_(3,4)=J_(4,5)=9.5 Hz, H-3_(E), 5_(E)), 3.85 (m, 2H,J_(3,4)=9.3, J_(4,5)=9.5 Hz, H-3_(A), 5_(A)), 3.72 (m, 2H, J_(5,6)=6.0Hz, H-4_(C), 5_(C)), 3.66-3.62 (m, 2H, H-6a_(E), 6b_(E)), 3.61 (dd, 1H,H-4_(E)), 3.54 (dd, 1H, J_(3,4)=9.4 Hz, H-3_(B)), 3.45 (dd, 1H,J_(4,5)=9.5, J_(5,6)=6.1 Hz, H-5_(B)), 3.39 (dd, 1H, H-2_(E)), 3.34 (dd,1H, H-4_(A)), 3.21 (dd, 1H, H-4_(B)), 1.89 (s, 3H, OAc), 1.26 (2d, 6H,H-6_(A), 6_(C)), 0.89 (d, 3H, H-6_(B)); ¹³C NMR: δ 170.2, 166.1 (C═O),138.4-118.1 (Ph, All), 101.3 (C-1_(B)), 99.8 (C-1_(A)), 98.2 (C-1_(E)),96.4 (C-1_(C)), 82.2 (C-3_(E)), 81.4 (C-2_(E)), 80.6 (C-4_(A)), 80.5(C-3_(C)), 80.1 (C-4_(B)), 79.3 (C-3_(B)), 78.5 (C-4_(C)), 78.1(C-3_(A)), 78.0 (C-4_(E)), 76.0, 75.9, 75.7, 75.2, 74.3, 73.3, 72.1,71.1 (8C, CH₂Ph), 75.2 (C-2_(B)), 72.9 (C-2_(C)), 71.7 (C-5_(E)), 69.5(C-2_(A)), 69.2 (2C, C-5_(A), 5_(B)), 68.9 (All), 68.9 (C-6_(E)), 67.9(C-5_(C)), 21.4 (OAc), 19.1 (C-6_(A)), 18.7 (C-6_(C)), 18.1 (C-6_(B)).FAB-MS for C₉₀H₁₀₀O₂₀ (M, 1524.7) m/z 1547.8 [M+Na]⁺. Anal. Calcd forC₉₂H₁₀₀O₂₀: C, 72.42; H, 6.61%. Found: C, 72.31; H, 6.75%.

Allyl(3,4-Di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(340). A mixture of 344 (6.0 g, 3.93 mmol) in MeOH (200 mL) was treatedwith 10 mL of HBF₄/Et₂O at rt. The solution was stirred during 5 days.Et₃N was added until neutralization and concentrated. The residue wasdiluted with DCM, washed with satd aq NaHCO₃ and water. The organiclayer was dried on MgSO₄, filtered and concentrated. The residue waseluted from a column of silica gel with 6:1 cyclohexane-EtOAc to give340 (5.0 g, 84%) as a colourless foam: [α]_(D)+12° (c 1.0, CHCl₃); ¹HNMR: δ 8.00-7.00 (m, 45H, Ph), 5.83 (m, 1H, All), 5.29 (dd, 1H,J_(1,2)=1.8, J_(2,3)=2.9 Hz, H-2_(C)), 5.19 (m, 2H, All), 4.99 (d, 1H,J_(1,2)=1.4 Hz, H-1_(A)), 4.97 (d, 1H, J_(1,2)=3.3 Hz, H-1_(E)), 4.94(d, 1H, J_(1,2)=1.7 Hz, H-1_(B)), 4.83 (d, 1H, H-1_(C)), 4.90-4.35 (m,16H, CH₂Ph), 4.30 (dd, 1H, J_(2,3)=2.7 Hz, H-2_(B)), 4.10-4.00 (m, 2H,All), 4.02 (dd, 1H, J_(2,3)=3.5, J_(3,4)=8.5 Hz, H-3_(C)), 3.98 (m, 1H,H-2_(A)), 3.95-3.91 (m, 3H, H-5_(E), 6a_(E), 6a_(E)), 3.90 (dd, 1H,J_(2,3)=9.5, J_(3,4)=9.4 Hz, H-3_(E)), 3.82-3.73 (m, 4H, H-3_(A), 5_(A),4_(C), 5_(C)), 3.66 (dd, 1H, J₄₅=9.6 Hz, H-4_(E)), 3.53 (dd, 1H,J_(3,4)=9.5 Hz, H-3_(B)), 3.48 (m, 1H, J_(4,5)=9.5 Hz, H-5_(B)),3.44-3.40 (m, 2H, H-4_(A), 2_(E)), 3.17 (pt, 1H, H-4_(B)), 2.18 (d, 1H,J_(2,OH)=2.0 Hz, OH), 1.26 (d, 3H, J_(5,6)=5.5 Hz, H-6_(C)), 1.25 (d,3H, J_(5,6)=6.2 Hz, H-6_(A)), 0.90 (d, 3H, J_(5,6)=6.2 Hz, H-6_(B)); ¹³CNMR: δ 166.2 (C═O), 138.3-118.0 (Ph, All), 101.5 (C-1_(B)), 101.4(C-1_(A)), 98.2 (C-1_(E)), 96.4 (C-1_(C)), 82.2 (C-3_(E)), 81.4(C-2_(E)), 80.6 (C-4_(A)), 80.3 (C-4_(B)), 79.9 (2C, C-3_(C), 3_(A)),79.2 (C-3_(B)), 78.3 (C-4_(C)), 78.0 (C-4_(E)), 75.9, 75.6, 75.5, 74.8,74.2, 73.5, 72.4, 71.0 (8C, CH₂Ph), 75.3 (C-2_(B)), 72.9 (C-2_(C)), 71.6(C-2_(A)), 69.2, 69.1, 68.3, 67.9 (4C, C-5_(A), 5_(B), 5_(C), 5_(E)),68.9, 68.7 (3C, C-6_(D), 6_(E), All), 19.1 (C-6_(C)), 18.6 (C-6_(A)),18.1 (C-6_(B)). FAB-MS for C₉₀H₉₈O₁₉ (M, 1482.7) m/z 1505.8 [M+Na]⁺.Anal. Calcd for C₉₀H₉₈O₁₉.2H₂O: C, 71.12; H, 6.77%. Found: C, 71.21; H,6.78%.

Allyl(3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(304). (a) A mixture of the donor 308 (200 mg, 230 μmol) and theacceptor 310 (188 mg, 144 μmol), 4 Å molecular sieves and dryEt₂O:1,2-DCE (1:1, 5 mL) was stirred for 1.5 h then cooled to 0° C. NIS(104 mg, 0.46 mmol) and triflic acid (4 μL, 0.05 mmol) were successivelyadded. The stirred mixture was allowed to reach rt in 1 h. Et₃N (25 μL)was added and the mixture filtered. After evaporation, the residue waseluted from a column of silica gel with 4:1 to 2:1 cyclohexane-EtOAc togive 304 (28 mg, 10%).

(b) A mixture of alcohol 310 (5.0 g, 3.37 mmol), imidate 316 (3.0 g,5.04 mmol) and 4 Å molecular sieves in anhydrous DCM (120 mL) wasstirred for 1 h under dry Argon. After cooling at 0° C., TMSOTf (240 μL,1.32 mmol) was added dropwise and the mixture was stirred for 2.5 hwhile coming back to rt. Et₃N (800 μL) was added, and the mixture wasfiltered and concentrated. The residue was eluted from a column ofsilica gel with 4:1 to 2:1 cyclohexane-EtOAc to give 304 (6.27 g, 98%)as a colourless foam: [α]_(D)+1.5° (c 1.0, CHCl₃); ¹H NMR: δ 8.00-7.00(m, 45H, Ph), 6.68 (d, 1H, J_(2,NH)=8.5 Hz, NH_(D)), 5.82 (m, 1H, All),5.29 (dd, 1H, J_(1,2)=1.0, J_(2,3)=2.3 Hz, H-2_(C)), 5.19 (m, 2H, All),5.00 (d, 1H, J_(1,2)=1.0 Hz, H-1_(A)), 4.96 (dd, 1H, J_(2,3)=10.5,J_(3,4)=10.5 Hz, H-3_(D)), 4.88 (d, 1H, J_(1,2)=3.3 Hz, H-1_(E)), 4.85(d, 1H, H-1_(C)), 4.82 (d, 1H, J_(1,2)=1.7 Hz, H-1_(B)), 4.81 (dd, 1H,J_(4,5)=10.0 Hz, H-4_(D)), 4.72 (d, 1H, J_(1,2)=8.6 Hz, H-1_(D)),4.90-4.35 (m, 16H, CH₂Ph), 4.38 (m, 1H, H-2_(B)), 4.10-4.00 (m, 2H,All), 4.05 (dd, 1H, J_(2,3)=2.7 Hz, H-2_(A)), 3.95 (dd, 1H, J_(2,3)=3.5,J_(3,4)=8.5 Hz, H-3_(C)), 3.90 (m, 2H, H-5_(E), 4_(E)), 3.86-3.82 (m,2H, H-6a_(D), 6b_(D)), 3.84-3.70 (m, 6H, H-3_(E), 6a_(E), 6b_(E), 3_(A),5_(A), 2_(D)), 3.68 (m, 1H, H-5_(C)), 3.61 (dd, 1H, J_(4,5)=9.0 Hz,H-4_(C)), 3.56 (dd, 1H, J_(3,4)=9.5 Hz, H-3_(B)), 3.47 (m, 1H,J_(4,5)=9.5, J_(5,6)=6.1 Hz, H-5_(B)), 3.35-3.33 (m, 3H, H-4_(A), 5_(D),2_(E)), 3.17 (dd, 1H, H-4_(B)), 2.02, 2.00, 1.98 (3s, 9H, OAc), 1.24 (d,3H, J_(5,6)=6.0 Hz, H-6_(A)), 1.23 (d, 3H, J_(5,6)=5.9 Hz, H-6_(C)),0.90 (d, 3H, H-6_(B)); ¹³C NMR: δ 170.9, 170.7, 169.6, 166.1, 162.1(C═O), 138.3-118.1 (Ph, All), 101.5 (C-1_(D)), 101.4 (C-1_(B)), 101.1(C-1_(A)), 98.5 (C-1_(E)), 96.4 (C-1_(C)), 92.6 (CCl₃), 82.1 (C-3_(E)),81.7 (C-3_(C)), 81.6 (C-2_(E)), 80.4 (C-4_(B)), 80.1 (C-3_(A)), 79.1(bs, C-4_(C)), 78.5 (C-3_(B)), 77.9 (C-4_(A)), 77.6 (C-4_(E)), 76.4(C-2_(A)), 76.1, 75.8, 75.4, 74.7, 74.3, 74.2, 73.2, 70.4 (8C, CH₂Ph),74.9 (C-2_(B)), 72.9 (C-3_(D)), 72.7 (C-2_(C)), 72.5 (C-5_(D)), 71.9(C-5_(E)), 68.4 (C-6_(E)), 68.8 (All), 68.9, 68.7, 68.5, 67.7 (4C,C-4_(D), 5_(A), 5_(B), 5_(C)), 62.1 (C-6_(D)), 56.2 (C-2_(D)), 20.9,20.7 (3C, OAc), 19.0 (C-6_(A)), 18.5 (C-6_(C)), 18.2 (C-6_(B)). FAB-MSof C₁₀₄H₁₁₄Cl₃NO₂₇ (M, 1916.4) m/z 1938.9 [M+Na]⁺. Anal. Calcd forC₁₀₄H₁₁₄Cl₃NO₂₇: C, 65.18; H, 6.00; N, 0.73%. Found: C, 64.95; H, 6.17;N, 0.76%.

(2,3,4-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranosyltrichloroacetimidate (346). Compound 304 (3.5 g, 1.8 mmol) was dissolvedin anhydrous THF (35 mL). The solution was degassed and placed underArgon. 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (81 mg) was added, and the solution was degassedagain. The catalyst was activated by passing over a stream of hydrogenuntil the solution has turned yellow. The reaction mixture was degassedagain and stirred under an Argon atmosphere, then concentrated todryness. The residue was dissolved in acetone (15 mL), then water (3mL), mercuric chloride (490 mg) and mercuric oxide (420 mg) were addedsuccessively. The mixture, protected from light, was stirred at rt for 2h and acetone was evaporated. The resulting suspension was taken up inDCM, washed twice with 50% aq KI, water and brine, dried andconcentrated. The residue was eluted from a column of silica gel with2:1 petroleum ether-EtOAc to give the corresponding hemiacetal 345.Trichloroacetonitrile (6.5 mL) and DBU (97 μL) were added to a solutionof the residue in anhydrous DCM (33 mL) at 0° C. After 1 h, the mixturewas concentrated. The residue was eluted from a column of silica gelwith 5:2 cyclohexane-EtOAc and 0.2% Et₃N to give 346 (2.48 g, 66%) as acolourless foam: [α]_(D)+4° (c 1.0, CHCl₃); ¹H NMR: δ 8.71 (s, 1H, NH),8.00-7.00 (m, 45H, Ph), 6.80 (d, 1H, J_(2,NH)=8.6 Hz, NH_(D)), 6.37 (d,1H, J_(1,2)=2.7 Hz, H-1_(C)), 5.59 (dd, 1H, J_(2,3)=2.9 Hz, H-2_(C)),5.10 (bs, 1H, H-1_(A)), 5.05 (pt, 1H, J_(2,3)=9.8 Hz, H-3_(D)),5.02-4.96 (m, 4H, H-1_(E), 1_(B), 4_(D), CH₂Ph), 5.00-4.42 (m, 17H, 15CH₂Ph, H-1_(D), 3_(C)), 4.14 (bs, 1H, H-2_(A)), 4.05-3.68 (m, 14H,H-3_(E), 4_(E), 5_(E), 6a_(E), 6b_(E), 4_(C), 5_(C), 2_(B), 3_(B),3_(A), 5_(A), 2_(D), 6a_(D), 6b_(D)), 3.61 (dq, 1H, J_(5,6)=6.2,J_(4,5)=9.3 Hz, H-5_(B)), 3.51-3.41 (m, 3H, H-2_(E), 4_(A), 5_(D)), 3.30(pt, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-4_(B)), 2.03, 2.02, 1.80 (3s, 9H,OAc), 1.39, 1.32 (2d, 6H, H-6_(A), 6_(C)), 1.00 (bd, 3H, H-6_(B)). ¹³CNMR: δ 169.7, 169.5, 168.3, 164.5, 160.9 (C═O, C═N), 137.5-126.2 (Ph),101.6 (C-1_(D)), 101.3 (2C, C-1_(A), 1_(B)), 98.7 (C-1_(E)), 94.8(C-1_(C)), 91.3 (CCl₃), 82.1, 81.5, 80.4, 80.1, 78.4, 77.9, 77.6, 76.5(10C, C-2_(A), 2_(E), 3_(A), 3_(B), 3_(C), 3_(E), 4_(A), 4_(B), 4_(C),4_(E)), 76.0, 75.9, 75.5, 74.9, 74.3, 73.3 (8C, CH₂Ph), 72.9, 72.6,71.9, 70.9, 70.6, 69.1, 68.8, 68.5 (9C, C-2_(B), 2_(C), 3_(D), 4_(D),5_(A), 5_(B), 5_(C), 5_(D), 5_(E)), 68.3 (C-6_(E)), 62.1 (C-6_(D)), 56.2(C-2_(D)), 21.0, 20.9, 20.8 (3C, OAc), 19.1, 18.3, 18.1 (3C, C-6_(A),6_(B), 6_(C)). Anal. Calcd for C₁₀₃H₁₁₀Cl₆N₂O₂₇: C, 61.22; H, 5.49; N,1.39%. Found: C, 61.24; H, 5.50; N, 1.21%.

Methyl(2-Deoxy-4,6-O-isopropylidene-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranosiden(348). The pentasaccharide 302 (578 mg, 0.321 mmol) was dissolved inMeOH (10 mL). MeONa was added until pH reach 10. The mixture was stirredfor 25 min then treated by IR 120 (H⁺) until neutral pH. The solutionwas filtered and concentrated. The residue was eluted from a column ofsilica gel with 9:1 DCM-MeOH to give the expected triol 347 (505 mg,89%). To a mixture of 347 (505 mg, 0.286 mmol) in dry DMF (2 mL) wasadded 2-methoxypropene (60 μL, 2.5 eq) and CSA (14 mg, cat). The mixturewas stirred 1 h and Et₃N (200 μL) was added. After evaporation, theresidue was eluted from a column of silica gel with 5:2cyclohexane-EtOAc with 0.3% of Et₃N to give 348 (420 mg, 81%) as acolorless foam: ¹H NMR: δ 8.00-7.00 (m, 45H, Ph), 7.17 (d, 1H, NH_(D)),5.39 (dd, 1H, J_(1,2)=1.2, J_(2,3)=3.0 Hz, H-2_(C)), 5.13 (d, 1H,J_(1,2)=1.1 Hz, H-1_(A)), 5.01 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)), 4.99(d, 1H, J_(1,2)=1.7 Hz, H-1_(B)), 4.80 (d, 1H, H-1_(C)), 4.70 (d, 1H,H-1_(D)), 4.90-4.35 (m, 16H, CH₂Ph), 4.40 (m, 1H, H-2_(B)), 4.10 (dd,1H, H-2_(A)), 4.05 (dd, 1H, H-3_(C)), 4.00-3.00 (m, 20H, H-4_(C), 5_(C),3_(B), 4_(B), 5_(B), 3_(A), 4_(A), 5_(A), 2_(D), 3_(D), 4_(D), 5_(D),6a_(D), 6b_(D), 2_(E), 3_(E), 4_(E), 5_(E), 6a_(E), 6b_(E)), 3.40 (s,3H, OCH₃), 1.40-1.00 (m, 15H, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)); ¹³C NMRpartial: δ 166.2, 164.4 (C═O), 137.5-126.5 (Ph), 101.8 (C-1_(D)), 101.4(C-1_(B)), 101.2 (C-1_(A)), 100.2 (C(CH₃)₂), 98.4 (C-1_(E)), 98.2(C-1_(C)), 92.4 (CCl₃), 68.5 (C-6_(E)), 61.8 (C-6_(D)), 60.1 (C-2_(D)),55.5 (OCH₃), 29.3, 19.4 (C(CH₃)₂), 19.1, 18.6, 18.2 (C-6_(A), 6_(B),6_(C)). FAB-MS of C₉₉H₁₁₀Cl₃N₁O₂₄ (M, 1804.1), m/z 1827.0 [M+Na]⁺. Anal.Calcd for C₉₉H₁₁₀Cl₃N₁O₂₄: C, 65.90; H, 6.15; N, 0.78%. Found: C, 65.89;H, 6.29; N, 0.68%.

Methyl(3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(350). A mixture of 346 (154 mg, 76 μmol) and 348 (92 mg, 51 μmol), 4 Åmolecular sieves and dry 1,2-DCE (3 mL), was stirred for 1 h, thencooled to −35° C. Triflic acid (6 μL) was added. The stirred mixture wasallowed to reach 10° C. in 2.5 h. Et₃N (25 μL) was added and the mixturewas filtered. After evaporation, the residue was eluted from a column ofsilica gel with 2:1 cyclohexane-EtOAc and 0.5% of Et₃N to give 349 (186mg) as a contaminated material. To a solution of the isolatedcontaminated 349 (186 mg) in DCM (3 mL) was added dropwise, at 0° C., asolution of TFA (0.5 mL) and water (0.5 mL). The mixture was stirred for3 h, then concentrated by co-evaporation with water then toluene. Theresidue was eluted from a column of silica gel with 2:1 to 1:1 petroleumether-EtOAc to give 350 (134 mg, 72%, 2 steps) as a white solid:[α]_(D)+6° (c 1.0, CHCl₃); ¹H NMR: δ 8.05-7.10 (m, 90H, Ph), 6.86-6.82(2d, 2H, J_(2,NH)=8.0, J_(2,NH)=8.5 Hz, NH_(D), NH_(D′)), 5.35-5.19 (m,2H, H-2_(C), 2_(C′)), 5.20, 5.08 (2s, 2H, H-1_(A), 1_(A′)), 5.05 (dd,1H, H-3_(D′)), 4.99-4.80 (m, 9H, H-1_(B), 1_(B′), 1_(C), 1_(C′), 1_(D),1_(D′), 1_(E), 1_(E′), 4_(D′)), 4.80-4.30 (m, 32H, OCH₂Ph), 4.10-3.15(m, 44H, H-2_(A), 2_(A′), 2_(B), 2_(B′), 2_(D), 2_(D′), 2_(E), 2_(E′),3_(A), 3_(A′), 3_(B), 3_(B′), 3_(C), 3_(C′), 3_(D), 3_(E), 3_(E′),4_(A), 4_(A′), 4_(B), 4_(B′), 4_(C), 4_(C′), 4_(D), 4_(E), 4_(E′),5_(A), 5_(A′), 5_(B), 5_(B′), 5_(C), 5_(C′), 5_(D), 5_(D′), 5_(E),5_(E′), 6a_(D), 6b_(D), 6a_(D′), 6b_(D′), 6a_(E), 6b_(E), 6a_(E′),6b_(E′)), 3.42 (3H, s, OMe), 2.08, 2.04, 2.02 (9H, 3s, OAc), 1.40-0.96(18H, m, H-6_(A), 6_(A′), 6_(B), 6_(B′), 6_(C), 6_(C′)); ¹³C NMR: δ171.5, 170.9, 170.8, 169.6, 166.2, 162.4, 162.1 (C═O), 139.5-127.2 (Ph),101.9, 101.6, 101.5, 101.3, 99.2, 98.8, 98.2 (10C, C-1_(A), 1_(A′),1_(B), 1_(B′), 1_(C), 1_(C′), 1_(D), 1_(D′), 1_(E), 1_(E′)), 92.7, 92.6(2C, CCl₃), 82.1, 81.8, 81.7, 80.5, 80.3, 80.1, 79.3, 77.9, 77.8, 73.0,72.6, 72.5, 72.0, 69.4, 69.0, 68.9, 67.4 (39C, C-2_(A), 2_(A′), 2_(B),2_(B′), 2_(C), 2_(C′), 2_(E), 2_(E′), 3_(A), 3_(A′), 3_(B), 3_(B′),3_(C), 3_(C′), 3_(D), 3_(D′), 3_(E), 3_(E′), 4_(A), 4_(A′), 4_(B),4_(B′), 4_(C), 4_(C′), 4_(D), 4_(D′), 4_(E), 4_(E′), 5_(A), 5_(A′),5_(B), 5_(B′), 5_(C), 5_(C′), 5_(D), 5_(D′), 5_(E), 5_(E′), 6_(D′)),76.0, 75.9, 74.8, 74.3, 73.6, 73.2, 68.6 (CH₂Ph), 62.3, 62.2, 60.7 (3C,C-6_(D), 6_(E), 6_(E′)), 55.5, 56.2 (3C, C-2_(D), 2_(D′), OCH₃), 21.0,20.9, 20.8 (OAc), 19.0, 18.7, 18.6, 18.2, 17.9 (6C, C-6_(A), 6_(A′),6_(B), 6_(B′), 6_(C), 6_(C′)). FAB-MS for C₁₉₇H₂₁₄Cl₆N₂O₅₀ (M, 3622.5)m/z 3645.3 [M+Na]⁺. Anal. Calcd for C₁₉₇H₂₁₄Cl₆N₂O₅₀: C, 65.32; H, 5.95;N, 0.77%. Found: C, 65.20; H, 6.03; N, 0.78%.

Methyl(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(α-L-rhamnopyranosyl)-(1→2)-(α-L-rhamnopyranosyl)-(1→3)-[α-D-glucopyranosyl-(1→4)]-(α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(α-L-rhamnopyranosyl)-(1→2)-(α-L-rhamnopyranosyl)-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranoside(301). A solution of 350 (183 mg, 50 μmol), in EtOH (3 mL), EtOAc (0.3mL), 1 M HCl (100 μL) was hydrogenated in the presence of Pd/C (250 mg)for 72 h at rt. The mixture was filtered and concentrated. A solution ofthe residue in MeOH (4 mL) and Et₃N (200 μL) was hydrogenated in thepresence of Pd/C (200 mg) for 24 h at rt. The mixture was filtered andconcentrated. A solution of the residue (50 mg, 25 μmol) in MeOH (3 mL)and DCM (0.5 mL) was treated by MeONa until pH reached 10. The mixturewas stirred overnight at 55° C. After cooling at rt, IR 120 (H⁺) wasadded until neutral pH, and the solution was filtered and concentrated,then was eluted from a column of C-18 with water/CH₃CN and freeze-driedto afford amorphous 301 (30 mg, 37%): [α]_(D)−1° (c 1.0, water); ¹H NMR(D₂O): δ 5.13 (2d, 2H, J_(1,2)=3.5 Hz, H-1_(E), 1_(E′)), 5.05, 4.95,4.75 (m, 5H, H-1_(A), 1_(B), 1_(A′), 1_(B′), 1_(C′)), 4.64-4.62 (2d, 2H,J_(1,2)=7.0, J_(1,2)=8.0 Hz, H-1_(D), 1_(D′)), 4.58 (d, 1H, J_(1,2)=2.2Hz, H-1_(C)), 4.10-3.20 (m, 51H, H-2_(A), 2_(A′), 2_(B), 2_(B′), 2_(C),2_(C′), 2_(D), 2_(D′), 2_(E), 2_(E′), 3_(A), 3_(A′), 3_(B), 3_(B′),3_(C), 3_(C′), 3_(D), 3_(D′), 3_(E), 3_(E′), 4_(A), 4_(A′), 4_(B),4_(B′), 4_(C), 4_(C′), 4_(D), 4_(D′), 4_(E), 4_(E′), 5_(A), 5_(A′),5_(B), 5_(B′), 5_(C), 5_(C′), 5_(D), 5_(D′), 5_(E), 5_(E′), 6a_(D),6b_(D), 6a_(D′), 6b_(D′), 6a_(E), 6b_(E), 6a_(E′), 6b_(E′), OCH₃), 1.99,1.97 (2s, 6H, 2 NHAc), 1.33-1.15 (6d, 18H, J_(5,6)=6.3 Hz, H-6_(A),6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′)); ¹³C NMR (D₂O): δ 175.2, 174.7(C═O), 103.1 (2C, C-1_(D′), 1_(D)), 102.6, 101.7, 101.3, 100.8 (6C,C-1_(A), 1_(B), 1_(C), 1_(A′), 1_(B′), 1_(C′)), 98.0 (2C, C-1_(E),1_(E′)), 81.6, 79.7, 79.6, 79.1, 76.2, 76.1, 73.9, 73.0, 72.7, 72.6,72.5, 72.2, 72.1, 71.6, 70.1, 70.0, 69.7, 69.0, 68.5 (38C, C-2_(A),2_(A′), 2_(B), 2_(B′), 2_(C), 2_(C′), 2_(E), 2_(E′), 3_(A), 3_(A′),3_(B), 3_(B′), 3_(C), 3_(C′), 3_(D), 3_(D′), 3_(E), 3_(E′), 4_(A),4_(A′), 4_(B), 4_(B′), 4_(C), 4_(C′), 4_(D), 4_(D′), 4_(E), 4_(E′),5_(A), 5_(A′), 5_(B), 5_(B′), 5_(C), 5_(C′), 5_(D), 5_(D′), 5_(E),5_(E′)), 60.9 (4C, C-6_(E), 6_(E′), 6_(D), 6_(D′)), 56.2, 56.0, 55.3(3C, C-2_(D), 2_(D′), OCH₃), 22.7, 22.6 (2C, NHAc), 18.3, 18.1, 17.2,17.1, 17.0, 16.9 (6C, C-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′)).HRMS (MALDI) calcd for [C₆₅H₁₁₀N₂O₄₅+Na]⁺: 1661.6278. Found: 1661.6277.

D—Synthesis of the 2-Amionoethyl Glycoside of a Hapten Representative ofthe O-Specific Polysaccharide of Shigella flexneri Serotype 2a and of aCorresponding PADRE-Conjugate

(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranosyltrichloroacetimidate (406).1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (25 mg, 29 μmol) was dissolved THF (5 mL), and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the colour to change toyellow. The solution was then degassed again in an argon stream. Asolution of 407 (1.0 g, 0.55 mmol) in THF (10 mL) was degassed andadded. The mixture was stirred at rt overnight, then concentrated todryness. The residue was dissolved in acetone (5 mL), then water (1 mL),mercuric chloride (140 mg) and mercuric oxide (120 mg) were addedsuccessively. The mixture protected from light was stirred at rt for 2 hand acetone was evaporated. The resulting suspension was taken up inDCM, washed twice with 50% aq KI, water and satd aq NaCl, dried andconcentrated. The residue was eluted from a column of silica gel with2:1 petroleum ether-EtOAc to give the corresponding hemiacetal 408.Trichloroacetonitrile (2.5 mL) and DBU (37 μL) were added to a solutionof the crude 408 in anhydrous DCM (12.5 mL) at 0° C. After 1 h, themixture was concentrated. The residue was eluted from a column of silicagel with 5:4 cyclohexane-EtOAc and 0.2% Et₃N to give 406 as a white foam(0.9 g, 85%); [α]_(D)+10° (c 1, CHCl₃). ¹H NMR: δ 8.70 (s, 1H, C═NH),8.00-7.00 (m, 45H, Ph), 6.36 (d, 1H, J_(1,2)=2.6 Hz, H-1_(C)), 5.59 (m,2H, N—H_(D), H-2_(C)), 5.13 (d, 1H, J_(1,2)=1.0 Hz, H-1_(A)), 5.01-4.98(m, 2H, H-1_(E), 1_(B)), 4.92 (dd, 1H, H-3_(D)), 4.90 (dd, 1H, H-4_(D)),4.68 (d, 1H, H-1_(D)), 5.00-4.02 (m, 19H, 8 CH₂Ph, H-3_(C), 2_(A),2_(B)), 4.01 (dd, 1H, H-2_(E)), 4.00-3.20 (m, 16H, H-3_(E), 4_(E),5_(E), 6a_(E), 6b_(E), 4_(C), 5_(C), 3_(B), 4_(B), 5_(B), 3_(A), 4_(A),5_(A), 5_(D), 6a_(D), 6b_(D)), 2.02, 2.00, 1.75, 1.65 (4s, 12H, C═OCH₃),1.40, 1.32 and 1.00 (3d, 9H, H-6_(A), 6_(B), 6_(C)). ¹³C NMR (partial):δ 170.2, 169.9, 169.3, 168.7, 164.9 (6C, C═O, C═N), 103.2 (C-1_(D)),101.4 (2C, C-1_(A), 1_(B)), 99.0 (C-1_(E)), 94.8 (C-1_(C)), 21.1, 20.9,20.8 (3C, CH₃C═O), 19.1, 18.2 (3C, C-6_(A), 6_(B), 6_(C)). Anal. Calcdfor C₁₀₃H₁₁₃Cl₃N₂O₂₇: C, 64.52; H, 5.94; N, 1.46%. Found: C, 64.47; H,5.99; N, 1.45%.

2-Azidoethyl(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(409). A mixture of alcohol 405 (110 mg, 330 μmol), trichloroacetimidate406 (720 mg, 376 μmol) and 4 Å molecular sieves in anhydrous 1,2-DCE (6mL) was stirred for 1 h under dry argon. After cooling at 0° C., TfOH(16 μL, 180 μmol) was added dropwise and the mixture was stirred at 80°C. for 2.5 h. Triethylamine (60 μL) was added and the mixture wasfiltered, and concentrated. The residue was eluted from a column ofsilica gel with 3:4 cyclohexane-EtOAc and Et₃N (0.2%) to give 409 as acolourless oil (540 mg, 78%); [α]_(D)+6.5° (c 1, CHCl₃). ¹H NMR: δ8.00-7.00 (m, 45H, Ph), 5.95 (d, 1H, J_(2,NH)=7.1 Hz, NH_(D)), 5.51 (d,1H, J_(2,NH)=8.1 Hz, NH_(D′)), 5.20 (dd, 1H, J_(1,2)=1.7, J_(2,3)=3.0Hz, H-2_(C)), 5.08 (d, 1H, J_(1,2)=1.0 Hz, H-1_(A)), 5.05 (d, 1H,J_(1,2)=8.3 Hz, H-1_(D)), 4.93 (d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.87(d, 1H, J_(1,2)=1.0 Hz, H-1_(B)), 4.82 (d, 1H, J_(1,2)=1.7 Hz, H-1_(C)),4.80 (dd, 1H, J_(3,4)=J_(4,5)=10.0 Hz, H-4_(D′)), 4.76 (dd, 1H,J_(2,3)=9.5 Hz, H-3_(D′)), 4.75-4.30 (m, 16H, CH₂Ph), 4.57 (d, 1H,J_(1,2)=7.8 Hz, H-1_(D′)), 4.35 (dd, 1H, H-2_(B)), 4.30 (dd, 1H,J_(2,3)=10.0, J_(3,4)=9.6 Hz, H-3_(D)), 4.02 (dd, 1H, J_(2,3)=2.0 Hz,H-2_(A)), 4.00-3.60 (m, 16H, H-6a_(D), 6b_(D), 3_(E), 4_(E), 5_(E),6a_(E), 6b_(E), 3_(C), 4_(C), 5_(C), 3_(B), 3_(A), 5_(A), 2_(D′),6a_(D′), 6b_(D′)), 3.48 (m, 1H, J_(4,5)=9.5 Hz, H-5_(B)), 3.46 (dd, 1H,H-4_(D)), 3.40 (m, 1H, H-5_(D)), 3.36 (dd, 1H, H-2_(E)), 3.35, 3.19 (m,4H, OCH₂CH₂N₃), 3.30 (dd, 1H, H-4_(A)), 3.19 (dd, 1H, J_(3,4)=9.5 Hz,H-4_(B)), 3.17 (m, 1H, H-5_(D)), 3.02 (m, 1H, H-2_(D)), 1.90-1.60 (6s,18H, CH₃C═O), 1.33, 1.26 (2s, 6H, C(CH₃)₂), 1.27 (d, 1H, J_(5,6)=6.2 Hz,H-6_(A)), 1.18 (d, 3H, J_(5,6)=6.1 Hz, H-6_(C)), 0.90 (d, 3H,J_(5,6)=6.1 Hz, H-6_(B)). ¹³C NMR: δ 172.1, 171.1, 170.8, 170.1, 169.6,166.2 (6C, C═O), 139.2-127.1 (Ph), 103.1A (C-1_(D′)), 101.6 (C-1_(B)),101.0 (C-1_(A)), 100.0 (C-1_(D)), 98.1 (C-1_(E)), 97.8 (C-1_(C)), 82.0(C-2_(E)), 81.7, 81.5, 80.2, 78.6, 78.4, 77.9, 77.9 (8C, C-3_(E), 4_(E),3_(C), 4_(C), 3_(B), 4_(B), 3_(A), 4_(A)), 77.8 (C-2_(A)), 76.0, 74.6(2C, C-3_(D), 3_(D′)), 74.0 (C-2_(B)), 73.4 (C-4_(D)), 73.3 (C-2_(C)),72.2, 71.9 (2C, C-5_(D), 5_(D′)), 68.9, 68.8, 67.7 (3C, C-5_(A), 5_(B),5_(E)), 68.6 (C-4_(D′)), 68.5 (C-6_(E)), 67.5 (C-5_(C)), 62.6, 62.2 (2C,C-6_(D), 6_(D′)), 59.7 (C-2_(D)), 54.6 (C-2_(D)), 51.0 (CH₂N₃), 29.5(C(CH₃)₂), 23.9, 23.5, 21.1, 20.9, 20.7 (5C, C═OCH₃), 19.6 (C(CH₃)₂),18.9 (C-6_(C)), 18.4 (C-6_(A)), 18.2 (C-6_(B)). FABMS of C₁₁₄H₁₃₃N₅O₃₂(M, 2085.3) m/z 2107.9 [M+Na]⁺

2-Azidoethyl(2,3,4-tri-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(410). To a solution of 409 (503 mg, 241 μmol) in AcOH (6 mL) was addedwater (1.5 mL) dropwise at rt. The mixture was stirred for 1 h at 60° C.then concentrated by successive co-evaporation with water and toluene.The residue was eluted from a column of silica gel with 1:4Cyclohexane-EtOAc to give 410 as a white foam (463 mg, 94%); [α]_(D)+9°(c 1, CHCl₃). ¹H NMR: δ 8.00-7.00 (m, 45H, Ph), 5.70 (d, 1H, NH_(D)),5.46 (d, 1H, J_(2,NH)=8.0 Hz, NH_(D′)), 5.25 (dd, 1H, H-2_(C)), 5.05 (d,1H, J_(1,2)=8.4 Hz, H-1_(D)), 5.00 (d, 1H, J_(1,2)=1.0 Hz, H-1_(A)),4.86 (m, 3H, H-1_(C), 3_(D′), 4_(D′)), 4.84 (m, 2H, H-1_(B), 1_(E)),4.56 (d, 1H, H-1_(D′)), 4.40 (dd, 1H, H-3_(E)), 4.35 (dd, 1H, H-2_(B)),4.15 (dd, 1H, H-3_(D)), 4.80-4.00 (m, 16H, CH₂Ph), 4.03 (dd, 1H,H-2_(A)), 4.00-3.00 (m, 26H, H-4_(D), 5_(D), 6a_(D), 6b_(D), 2_(E),4_(E), 5_(E), 6a_(E), 6b_(E), 3_(C), 4_(C), 5_(C), 3_(B), 4_(B), 5_(B),3_(A), 4_(A), 5_(A), 2_(D′), 5_(D′), 6a_(D′), 6b_(D′), OCH₂CH₂N₃), 2.99(m, 1H, H-2_(D)), 1.85-1.60 (5s, 15H, CH₃C═O), 1.25 and 0.85 (3d, 9H,H-6_(A), 6_(B), 6_(C)). ¹³C NMR (partial): δ 171.6, 171.4, 170.8, 170.1,169.6 (C═O), 140.0-127.1 (Ph), 103.1 (C-1_(D′)), 101.2 (C-1_(A)), 99.6(2C, C-1_(E), 1_(B)), 99.4 (C-1_(D)), 99.0 (C-1_(C)), 23.8, 23.5 (2C,NHAc), 21.1, 20.9, 20.8 (3C, OAc), 19.1, 18.5, 18.2 (C-6_(A), 6_(B),6_(C)). FABMS of C₁₁₁H₁₂₉N₅O₃₂ (M, 2045.2), m/z 2067.9 [M+Na]⁺. Anal.Calcd for C₁₁₁H₁₂₉N₅O₃₂: C, 65.19; H, 6.36; N, 3.42%. Found: C, 65.12;H, 6.51; N, 3.41%.

2-Aminoethyl(2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1→2)-(α-L-rhamnopyranosyl)-(1→2)-(α-L-rhamnopyranosyl)-(1→3)-[α-D-glucopyranosyl-(1→4)]-(α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(402). A solution of 410 (207 mg, 101 μmol) in MeOH (5 mL) was treatedby MeONa until pH 9. The mixture was stirred 1 week at rt. IR 120 (H⁺)was added until neutral pH and the solution was filtered, andconcentrated. The residue was eluted from a column of silica gel with20:1 to 15:1 DCM-MeOH to give amorphous 411. A solution of crude 411 inEtOH (2.2 mL), EtOAc (220 μL), 1 M HCl (172 μL, 2 eq) was hydrogenatedin the presence of Pd/C (180 mg) for 72 h at rt. The mixture wasfiltered and concentrated. Elution of the residue from a column of C18with water and freeze-drying of appropriate fractions resulted inamorphous 402 (81 mg, 77%); [α]_(D)−10° (c 1, water). ¹H NMR partial(D₂O): δ 5.12 (d, 1H, J_(1,2)=3.4 Hz, H-1_(E)), 5.07 (d, 1H, J_(1,2)=1.0Hz, H-1_(Rha)), 4.94 (d, 1H, J_(1,2)=10 Hz, H-1_(Rha)), 4.75 (d, 1H,J_(1,2)=1.0 Hz, H-1_(Rha)), 4.63 (d, 1H, J_(1,2)=8.35 Hz, H-1_(GlcNac)),4.54 (d, 1H, J_(1,2)=8.3 Hz, H-1_(GlcNac)), 1.98 and 1.96 (2s, 6H, 2CH₃C═ONH), 1.28-1.20 (m, 9H, H-6_(A), 6_(B), 6_(C)). ¹³C NMR partial(D₂O): δ 175.2, 174.8 (2C, C═O), 103.1 (C-1_(D′)), 101.6, 101.4 (3C,C-1_(A), 1_(B), 1_(C)), 100.8 (C-1_(D)), 97.9 (C-1_(E)), 56.2, 55.4 (2C,C-2_(D), 2_(D′)), 22.7, 22.6 (2 NHAc), 18.2, 17.2, 17.0 (3C, C-6_(A),6_(B), 6_(C)). HRMS (MALDI)Calcd for C₄₂H₇₃N₃O₂₈Na: 1090.4278. Found1090.4286.

(S-Acetylthiomethyl)carbonylaminoethyl2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(412). A solution of 404 (3.4 mg, 11.4 μmol) in CH₃CN (50 μL) was addedto the aminoethyl hexasaccharide 402 (4.1 mg, 3.84 μmol) in 0.1 Mphosphate buffer (pH 7.4, 500 μL). The mixture was stirred at rt for 1 hand purified by RP-HPLC to give 412 (2.7 mg, 59%). HPLC (230 nm): Rt14.27 min (99.9% pure, Kromasil 5 μm C18 100 Å 4.6×250 mm analyticalcolumn, using a 0-20% linear gradient over 20 min of CH₃CN in 0.01 M aqTFA at 1 mL/min flow rate). ES-MS for C₄₆H₇₇N₃O₃₀S (M, 1184.19) m/z1184.08.

PADRE (thiomethyl)carbonylaminoethyl2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(401). Compound 412 (4.9 mg, 4.12 μmol) was dissolved in water (350 μL)and added to a solution of 403 (9.1 mg, 5.2 μmol) in a mixture of water(750 μL), CH₃CN (150 μL) and 0.5 M phosphate buffer (pH 5.6, 900 μL). 89μL of a solution of hydroxylamine hydrochloride (139 mg/mL) in 0.5 Mphosphate buffer (pH 5.6) was added and the mixture was stirred for 2 h.RP-HPLC purification gave the pure target 401 (6.3 mg, 53%). HPLC (230nm): Rt 9.70 min (100% pure, Kromasil 5 μm C18 100 Å 4.6×250 mmanalytical column, using a 20-50% linear gradient over 20 min of CH₃CNin 0.01 M aq TFA at 1 mL/min flow rate). ES-MS Calcd for C₁₅₃H₂₅₄N₂₄O₆₅S(M, 2901.34) m/z 2901.20.

E—Preparation of Chemically Defined Glycopeptides as Potential SyntheticConjugate Vaccines Against Shigella flexneri Serotype 2a Disease

Solvent mixtures of appropriately adjusted polarity used forchromatography consisted of A, dichloromethane-methanol; B,cyclohexane-ethyl acetate, C, cyclohexane-acetone, D, toluene-ethylacetate.

2-Azidoethyl2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside (507).Camphorsulfonic acid (200 mg, 0.9 mmol) was added to a solution of triol514 (1.31 g, 4.52 mmol) in a mixture of DMF (4 mL) and2,2-dimethoxypropane (4 mL). After 3 h at rt, low boiling point solventswere evaporated under reduced pressure and more 2,2-dimethoxypropane (2mL, 15.8 mmol) was added. The mixture was stirred for 2 h at rt, Et₃Nwas added, and the mixture was concentrated. The crude product waspurified by column chromatography (solvent A, 19:1) to give 507 as awhite solid (1.21 g, 81%), [α]_(D)−89.8; ¹H NMR: δ 6.15 (d, 1H, J=5.9Hz, NH), 4.70 (d, 1H, J_(1,2)=8.3 Hz, H-1), 4.05 (m, 1H, OCH₂),3.97-3.89 (m, 2H, H-6a, 3), 3.79 (pt, 1H, J_(5,6b)=J_(6a,6b)=10.5 Hz,H-6b), 3.70 (m, 1H, OCH₂), 3.62-3.46 (m, 3H, H-2, 4, OCH₂), 3.35-3.26(m, 2H, H-5, CH₂N₃), 2.05 (s, 3H, Ac), 1.52 (s, 3H, C(CH₃)₂), 1.44 (s,3H, C(CH₃)₂); ¹³C NMR: δ 100.9 (C-1), 74.3 (C-4), 81.8 (C-3), 68.6(OCH₂), 67.3 (C-5), 62.0 (C-6), 58.7 (C-2), 50.7 (CH₂N₃), 29.0(C(CH₃)₂), 23.6 (CH₃CO), 19.1 (C(CH₃)₂). CIMS for C₁₃H₂₂N₄O₆ (330) m/z331 [M+H]⁺. Anal. Calcd. for C₆₇H₇₄N₄O₁₇.0.5H₂O: C, 46.01; H, 6.83; N,16.51%. Found C, 46.37; H, 6.69; N, 16.46%.

2-Azidoethyl(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(515) and 2-Azidoethyl(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(516). (a) The disaccharide donor 504 (1.425 g, 1.37 mmol) and theacceptor 507 (377 mg, 1.14 mmol) with 4 Å-MS (2 g) were placed underargon and CH₂Cl₂ (15 mL) was added. The mixture was stirred for 1 h atrt, then cooled to −40° C. A solution of BF₃.OEt₂ (0.5 mL, 4.11 mmol) inCH₂Cl₂ (5 mL) was added dropwise. The mixture was stirred at −40° C. to−15° C. over 3 h. Triethylamine (2.5 mL) was added and the mixturestirred for 20 min. The mixture was filtered through a pad of Celite,and the filtrate was concentrated. The mixture was purified by columnchromatography (solvent B, 2:3) to give 515 (803 mg, 58%) as acolourless foam. Further elution (solvent A, 9:1) gave 516 (395 mg, 30%)as a colourless foam. Compound 516 had [α]_(D)+91.5 (c 0.18); ¹H NMR: δ6.99-8.02 (m, 30H, Ph), 6.10 (d, 1H, J_(NH,2)=6.9 Hz, NH), 5.60 (dd, 1H,J_(2,3)=3.4, J_(3,4)=9.1 Hz, H-3_(C)), 5.52 (dd, 1H, H-2_(C)), 5.20 (d,1H, J_(1,2)=8.3 Hz, H-1_(D)), 5.00 (d, 1H, J_(1,2)=1.9 Hz, H-1_(C)),4.95 (d, 1H, J_(1,2)=3.4 Hz, H-1_(E)), 4.89-4.63 (m, 5H, CH₂Ph), 4.47(dd, 1H, J_(2,3)=8.3, J_(3,4)=10.3 Hz, H-3_(D)), 4.25 (d, 1H, J=10.9 Hz,CH₂Ph), 4.19 (m, 2H, H-5_(C), CH₂Ph), 4.06 (m, 1H, CH₂O), 3.87 (m, 5H,H-3_(E), 4_(C), 6a_(D), 6b_(D), CH₂Ph), 3.74-3.58 (m, 4H, H-4_(E),5_(D), 5_(E), CH₂O), 3.50 (m, 3H, H-2_(E), 4_(D), CH₂N₃), 3.32 (d, 1H,J_(6a,6b)=9.6 Hz, H-6a_(E)), 3.26 (m, 1H, CH₂N₃), 3.04 (d, 2H, H-2_(D),6b_(E)), 2.02 (s, 3H, CH₃CO), 1.51 (d, 3H, J_(5,6)=6.2 Hz, H-6_(C)); ¹³CNMR: δ 171.5, 165.6, 165.2 (3C, C═O), 138.6-127.3 (Ph), 99.6 (C-1_(C)),99.5 (C-1_(E)), 99.0 (C-1_(D)), 83.4 (C-3_(D)), 81.6 (C-3_(E)), 80.1(C-2_(E)), 79.2 (C-4_(C)), 77.2 (C-4_(E)), 75.5 (CH₂Ph), 75.1 (C-4_(D)),74.7, 74.0, 73.2 (3C, CH₂Ph), 71.3 (C-5_(D)*), 70.9 (C-5_(E)*), 70.8(C-3_(C)), 70.4 (C-2_(C)), 69.0 (C-5_(C)), 68.8 (CH₂O), 67.5 (C-6_(E)),62.6 (C-6_(D)), 57.9 (C-2_(D)), 50.5 (CH₂N₃), 23.4 (CH₃CO), 18.2(C-6_(C)). FAB-MS for C₆₄H₇₀N₄O₁₇ (M, 1166) m/z 1185 [M+Na]⁺. Anal.Calcd. for C₆₄H₇₀N₄O₁₇.H₂O: C, 64.85; H, 6.12; N, 4.73%. Found: C,64.71; H, 6.01; N, 4.83%.

(b) 4 Å Molecular sieves (560 mg) were added to a solution of donor 504(565 mg, 0.54 mmol) and acceptor 507 (150 mg, 0.45 mmol) in DCM (3 mL)and the suspension was stirred for 15 min −40° C. Triflic acid (16 μL)was added and the mixture was stirred for 3 h at rt once the coolingbath had reached rt. Et₃N was added and after 15 min, the mixture wasfiltered through a pad of Celite. Volatiles were evaporated and theresidue was column chromatographed (solvent B, 9:1) to give 515 (475 mg,87%). [α]_(D)+87.7 (c 0.32); ¹H NMR: δ 8.07-6.99 (m, 30H, Ph), 6.21 (d,1H, NH), 5.58 (dd, 1H, H-3_(C)), 5.44 (m, 1H, H-2_(C)), 5.13 (d, 1H,J_(1,2)=8.3 Hz, H-1_(D)), 5.02 (d, 1H, J_(1,2)=3.4 Hz, H-1_(E)), 4.97(d, 1H, J_(1,2)=1.5 Hz, H-1_(C)), 4.64-4.90 (m, 5H, CH₂Ph), 4.45 (t, 1H,H-3_(D)), 4.27 (m, 3H, H-5_(C), CH₂Ph), 4.05-3.79 (m, 7H, H-3_(E),4_(C), 5_(D), 6a_(D), 6b_(D), CH₂O, CH₂Ph), 3.60-3.76 (m, 4H, H-4_(D),4_(E), 5_(E), CH₂O), 3.37-3.51 (m, 3H, H-2_(E), 5_(D), CH₂N₃), 3.34-3.16(m, 3H, H-2_(D), 6a_(E), CH₂N₃), 3.04 (d, 1H, H-6b_(E)), 2.01 (s, 3H,CH₃C═O), 1.43 (s, 6H, (CH₃)₂C), 1.36 (d, 3H, H-6_(C)); ¹³C NMR: δ 171.7,165.6, 163.4 (C═O), 138.6-127.3 (Ph), 99.6 (C-1_(D)), 99.1 (C-1_(E)),97.7 (C-1_(C)), 91.9 ((CH₃)₂C), 81.4 (C-3_(E)), 80.3 (C-2_(E)), 79.4(C-4_(C)), 77.1 (C-4_(D)), 76.0 (C-3_(D)), 75.3, 74.6, 73.9, 73.2 (4C,CH₂Ph), 73.1 (C-4_(E)), 71.2 (2C, C-2_(C), 3_(C)), 71.1 (C-5_(E)), 68.6(CH₂O), 67.5 (C-5_(C)), 67.4 (C-6_(E)), 67.1 (C-5_(D)), 62.1 (C-6_(D)),59.0 (C-2_(D)), 50.5 (CH₂N₃), 28.9 ((CH₃)₂C), 23.4 (CH₃CO), 19.2((CH₃)₂C), 18.1 (C-6_(C)). FAB-MS for C₆₇H₇₄N₄O₁₇ (1206) m/z 1229[M+Na]⁺. Anal. Calcd. for C₆₇H₇₄N₄O₁₇: C, 60.41; H, 5.66; N, 4.82%.Found: C, 60.36; H, 5.69; N, 4.78%.

2-Azidoethyl(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(517). An ice cold solution of 95% aq TFA (1.5 mL) in CH₂Cl₂ (13.5 mL)was added to the trisaccharide 515 (730 mg, 0.60 mmol). The mixture waskept at 0° C. for 15 min, then diluted with toluene and concentrated.Toluene was co-evaporated from the residue. The residue was dissolved inMeOH (20 mL), and a 1M solution of sodium methoxide in MeOH (1.5 mL) wasadded. The mixture was left to stand at rt for 3 h. The mixture wasneutralised with Amberlite IR-120 (H⁺) resin and filtered. The filtratewas concentrated. The mixture was purified by column chromatography(solvent A, 9:1) to give 517 (548 mg, 94%) as a colourless foam.[α]_(D)+9.7 (c 0.48, MeOH); ¹H NMR: δ 7.13-7.31 (m, 8H, Ph), 5.99 (d,1H, J_(NH,2)=7.8 Hz, NH), 4.97-4.79 (m, 7H, H-1_(C), 1_(D), 1_(E),CH₂Ph), 4.374-4.35 (m, 4H, CH₂Ph), 4.10-3.91 (m, 7H, H-2_(C), 3_(D),3_(E), 5_(C), 5_(E), 6a_(D), CH₂O), 3.80 (m, 2H, H-3_(E), 6b_(D)), 3.73(m, 1H, CH₂O), 3.40-3.63 (m, 8H, H-2_(E), 4_(C), 4_(D), 4_(E), 5_(D),6a_(E), 6b_(E), CH₂N₃), 3.27 (m, 2H, H-2_(D), CH₂N₃), 1.99 (s, 3H,CH₃CO), 1.41 (d, 3H, J_(5,6)=6.2 Hz, H-6_(C)); ¹³C NMR: δ 170.7 (C═O),138.4-127.6 (Ph), 101.2 (C-1_(C)), 99.7 (C-1_(E)), 99.0 (C-1_(D)), 84.7(C-4_(C)), 84.3 (C-3_(D)), 81.5 (C-3_(E)), 79.6 (C-2_(E)), 77.6(C-4_(D)*), 75.6 (CH₂Ph), 75.3 (C-4_(E)*), 74.9, 73.5, 73.4 (3C, CH₂Ph),71.2 (C-5_(E)), 70.8 (C-5_(C)), 70.8 (C-5_(D)), 69.4 (C-3_(C)), 68.6(C-6_(E)), 68.4 (CH₂O), 67.6 (C-2_(C)), 62.6 (C-6_(D)), 56.4 (C-2_(D)),50.5 (CH₂N₃), 23.5 (CH₃CO), 17.6 (C-6_(C)). FAB-MS for C₅₀H₆₂N₄O₁₅ (958)m/z 981 [M+Na]⁺. Anal. Calcd. for C₅₀H₆₂N₄O₁₅.H₂O: C, 61.46; H, 6.60; N,5.73%. Found: C, 61.41; H, 6.61; N, 5.97%.

2-Aminoethylα-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(518). The trisaccharide 517 (368 mg, 0.38 mmol) was dissolved in amixture of EtOH (10 mL) and EtOAc (1 mL). A 1N solution of aqueous HCl(0.77 mL) was added. The mixture was stirred under hydrogen in thepresence of 10% Pd/C (400 mg) for 24 h. The mixture was diluted withwater and filtered. The filtrate was concentrated, then lyophilised. Theresidue was dissolved in a solution of NaHCO₃ (75 mg) in water (1 mL)and purified by passing first through a column of C₁₈ silica (elutingwith water), then through a column of Sephadex G₁₀ (eluting with water)to give, after lyophilisation, 518 (151 mg, 69%). HPLC (215 nm): Rt 4.09min (Kromasil 5 μm C18 100 Å 4.6×250 mm analytical column, using a 0-20%linear gradient over 20 min of CH₃CN in 0.01M aq TFA at 1 mL/min flowrate). ¹H NMR (D₂O): δ 4.97 (d, 1H, J_(1,2)=3.8 Hz, H-1_(E)), 4.78 (d,1H, J_(1,2)=1.2 Hz, H-1_(C)), 4.54 (d, 1H, J_(1,2)=8.6 Hz, H-1_(D)),4.02 (m, 1H, H-5_(C)), 5.00-3.90 (m, 3H, H-5_(E), 6a_(D), CH₂O),3.88-3.67 (m, 7H, H-2_(C), 2_(D), 3_(C), 6a_(E), 6b_(E), 6b_(D), CH₂O),3.61 (dd, 1H, J=9.8, J=9.1 Hz, H-3_(E)), 3.60-3.42 (m, 5H, H-2_(E),4_(C), 4_(D), 4_(E), 5_(D)), 3.54 (m, 1H, H-3_(D)), 3.03 (m, 2H,CH₂NH₂), 2.00 (s, 3H, CH₃CO), 1.31 (d, 3H, J_(5,6)=6.3 Hz, H-6_(C)); ¹³CNMR (D₂O): δ 175.2 (C═O), 101.6 (C-1_(C)), 100.7 (C-1_(D)), 100.0(C-1_(E)), 82.1 (C-3_(D)), 81.4 (C-4_(C)), 76.3 (C-2_(E)), 73.1(C-3_(E)), 72.2 (C-5_(E)), 71.9 (C-4_(D)), 71.3 (C-2_(C)), 69.7(C-4_(E)), 69.3 (C-3_(C)), 68.8 (C-5_(D)), 68.5 (C-5_(C)), 66.0 (CH₂O),60.9 (C-6_(D)), 60.5 (C-6_(E)), 55.5 (C-2_(D)), 39.8 (CH₂NH₂), 22.57(CH₃CO), 17.1 (C-6_(C)). ES-MS for C₂₂H₄₀N₂O₁₅ (572) m/z 573 [M+H]⁺.HRMS (MALDI) Calcd for C₂₂H₄₀N₂O₁₅Na: 595.2326. Found: 595.2341.

Allyl(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(521). TMSOTf (100 μL) was added to a solution of donor 520 (2.5 g, 5.78mmol) and acceptor 519 (4.0 g, 4.80 mmol) in Et₂O (40 mL) at −50° C. Themixture was stirred for 2.5 h, at which time the cooling bath hadreached rt. Et₃N was added and after 15 min, volatiles were evaporated.Column chromatography (solvent C, 4:1) of the crude product gave thefully protected 521 (4.74 g, 89%) as a white solid. ¹H NMR: δ 8.00-6.90(m, 25H, Ph), 5.92 (m, 1H, CH═), 5.53 (dd, 1H, H-2_(B)), 5.40-5.20 (m,4H, H-1_(E), 2_(C), CH₂═), 5.18 (dd, 1H, J_(2,3)=3.4, J_(3,4)=10.0 Hz,H-3_(B)), 5.10 (d, 1H, J_(1,2)=1.6 Hz, H-1_(B)), 5.00-4.40 (m, 10H,H-4_(B), 1_(C), OCH₂), 4.30-4.00 (m, 5H, H-3_(E), 3_(C), 5_(E), OCH₂),4.00-3.50 (m, 7H, H-2_(E), 4_(E), 6a_(E), 6b_(E), 5_(B), 5_(C), 4_(C)),1.90 (s, 3H, Ac), 1.60 (s, 3H, Ac), 1.22 (s, 3H, Ac), 1.20 (d, 3H,J_(5,6)=6.2 Hz, H-6_(C)), 0.80 (d, 3H, J_(5,6)=6.2 Hz, H-6_(B)); ¹³CNMR: δ 169.9, 169.7, 169.5, 166.1 (4C, C═O), 133.4-127.3 (Ph), 117.5(═CH₂), 9.8 (C-1_(B)), 96.9 (C-1_(E)), 95.7 (C-1_(C)), 81.4 (C-3_(E)),80.7 (C-2_(E)), 7.3 (C-3_(C)), 77.7 (C-4_(E)), 77.5 (brs, C-4_(C)),75.3, 74.6, 73.6 (3C, OCH₂Ph), 72.8 (C-2_(C)), 72.6 (CH₂Ph), 70.9 (2C,C-5_(E), 4_(B)), 69.6 (C-2_(B)), 68.7 (C-6_(E)), 68.6 (C-3_(B)), 68.2(OCH₂), 67.2 (C-5_(C)), 66.8 (C-5_(B)), 20.7, 20.3, 20.2 (3C, C(O)CH₃),18.5 (C-6_(C)), 16.8 (C-6_(B)). CI-MS for C₆₂H₇₀O₁₈ (1102) m/z 1125[M+Na]⁺. Anal. Calcd. for C₆₂H₇₀O₁₈: C, 67.50; H, 6.40%. Found: C,67.51; H, 6.52%.

(2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-2-O-benzoyl-α-L-rhamnopyranose(522). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (33 mg) was dissolved in THF (10 mL) and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, until the colour had changed toyellow. The solution was then degassed again in an argon stream. Asolution of 521 (4.59 g, 4.16 mmol) in THF (30 mL) was degassed andadded. The mixture was stirred at rt overnight, then concentrated. Theresidue was taken up in a mixture of acetone (10:1, 44 mL). Mercuricbromide (1.78 g, 8.32 mmol) and mercuric oxide (1.69 g, 6.24 mmol) wereadded to the mixture, which was protected from light. The suspension wasstirred at rt for 1 h, then concentrated. The residue was taken up inCH₂Cl₂ and washed three times with sat aq KI, then with brine. Theorganic phase was dried and concentrated. The residue was purified bycolumn chromatography (solvent B, 3:1) to give 522 (3.52 g, 80%) as acolourless foam; ¹H NMR: δ 7.15 (m, 25H, Ph), 5.50 (dd, 1H, H-2_(B)),5.30-5.27 (m, 2H, H-1_(C), H-2_(C)), 5.23 (d, 1H, J_(1,2)=3.3 Hz,H-1_(E)), 5.18 (dd, 1H, J_(2,3)=3.2, J_(3,4)=10.0 Hz, H-3_(B)), 5.10 (d,1H, J_(1,2)=1.2 Hz, H-1_(B)), 5.00-4.35 (m, 9H, H-4_(B), OCH₂), 4.28(dd, 1H, J_(2,3)=3.2, J_(3,4)=8.6 Hz, H-3_(C)), 4.20-4.00 (m, 3H,H-3_(E), 5_(E), 5_(C)), 3.80-3.50 (m, 6H, H-2_(E), 6a_(E), 6b_(E),5_(B), 4_(E), 4_(C)), 3.05 (d, 1H, J_(OH,1)=4.0 Hz, OH), 2.09, 1.81,1.44 (3s, 9H, CH₃C═O), 1.37 (d, 3H, J_(5,6)=6.2 Hz, H-6_(C)), 0.95 (d,3H, J_(5,6)=6.2 Hz, H-6_(B)); ¹³C NMR: δ 169.9, 169.8, 169.6, 166.2 (4C,C═O), 138.9-127.5 (Ph), 99.8 (C-1_(B)), 97.3 (C-1_(E)), 91.3 (C-1_(C)),81.7 (C-3_(E)), 80.7 (C-2_(E)), 78.8 (C-3_(C)), 78.1, 78.0 (2C, C-4_(E),4_(C)), 76.6, 75.5 (2C, CH₂Ph), 74.9 (2C, C-2_(E), CH₂Ph), 73.8 (CH₂Ph),73.3 (2C, C-4_(B), 5_(E)), 72.9 (C-2_(B)), 71.2 (2C, C-3_(B), 6_(E)),67.5 (C-5_(C)), 67.1 (C-5_(B)), 21.0, −20.6, 20.5 (3C, CH₃C═O), 18.9(C-6_(C)), 17.1 (C-6_(B)). FAB-MS for C₅₉H₆₆O₁₈ (1062) m/z 1085 [M+Na]⁺.Anal. Calcd. for C₅₉H₆₆O₁₈.H₂O: C, 65.54; H, 6.34%. Found: C, 65.68; H,6.41%.

(2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-2-O-benzoyl-α-L-rhamnopyranosetrichloroacetimidate (505). DBU (100 μL) was added at 0° C. to asolution of the hemiacetal 522 (3.8 g, 3.58 mmol) in DCM (40 mL)containing trichloroacetonitrile (4 mL). The mixture was stirred for 30min at 0° C., and volatiles were evaporated. Flash chromatography(solvent B, 7:3+0.2% Et₃N) of the crude material gave the donor 505 (3.9g, 90%) as a white solid; ¹H NMR (a anomer): δ 8.75 (s, 1H, NH),8.13-7.12 (m, 25H, Ph), 6.40 (d, 1H, J_(1,2)=2.4 Hz, H-1_(C)), 5.54 (brs, 1H, H-2_(B)), 5.49 (dd, 1H, J_(2,3)=2.9 Hz, H-2_(C)), 5.26 (d, 1H,J_(1,2)=2.8 Hz, H-1_(E)), 5.20 (dd, 1H, J_(2,3)=J_(3,4)=10.0 Hz,H-3_(B)), 5.17 (br s, 1H, H-1_(B)), 4.96 (dd, 1H, H-4_(B)), 4.99-4.41(m, 8H, OCH₂), 4.34 (m, 1H, H-3_(C)), 4.14-4.02 (m, 3H, H-3_(E), 5_(E),5_(C)), 3.87 (m, 1H, H-4_(C)), 3.78 (dq, 1H, J_(4,5)=9.5, J_(5,6)=6.1Hz, H-5_(B)), 3.70 (m, 2H, H-6a_(E), 6b_(E)), 3.65 (dd, 1H, J_(2,3)=3.4,J_(3,4)=9.8 Hz, H-2_(E)), 3.57 (pt, 1H, J_(2,3)=J_(3,4)=9.4 Hz,H-4_(E)), 1.86, 1.83 (2s, 9H, CH₃CO), 1.42 (d, 3H, J_(5,6)=6.2 Hz,H-6_(C)), 0.98 (d, 3H, H-6_(B)); ¹³C NMR (α anomer): δ 170.3, 170.1,169.9, 166.1 (4C, C═O), 160.7 (C═NH), 139.2-127.8 (Ph), 100.2 (C-1_(B)),98.1 (C-1_(E)), 94.8 (C-1_(C)), 91.2 (CCl₃), 82.4 (C-4_(C)), 82.0(C-3_(E)), 81.2 (br s, C-2_(E)), 78.5 (br s, C-3_(C)), 78.3 (C-4_(E)),75.9, 75.4, 74.3, 73.3 (4C, CH₂Ph), 71.8 (C-5_(E)), 71.7 (C-2_(C)), 71.3(C-4_(B)), 70.8 (br s, C-5_(C)), 70.0 (C-2_(B)), 69.3 (C-3_(B)), 69.2(C-6_(E)), 67.6 (C-5_(B)), 21.3, 21.0, 20.9 (3C, CH₃CO), 18.9 (C-6_(C)),17.1 (C-6_(B)). Anal. Calcd. for C₆₁H₆₆Cl₃NO₁₈: C, 60.67; H, 5.51; N,1.16%. Found: C, 60.53; H, 5.48; N, 1.38%.

2-Azidoethyl(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(523). The trisaccharide donor 505 (1.86 g, 1.54 mmol) and the acceptor507 (712 mg, 2.16 mmol) were dissolved in 1,2-dichloroethane (15 mL) and4 Å-MS (2 g) were added. The mixture was stirred at rt for 1 h. Themixture was cooled to 0° C. and triflic acid (34 μL, 0.385 mmol) wasadded. The mixture was stirred at 0° C. for 30 min, then at rt for 30min. The mixture was then heated at 65° C. for 1 h. The mixture wasallowed to cool, Et₃N (0.5 mL) was added, and the mixture was stirred atrt for 20 min. The mixture was diluted with CH₂Cl₂ and filtered througha pad of Celite. The filtrate was concentrated and purified by columnchromatography (solvent B, 1:1) to give 523 (1.61 g, 76%). ¹H NMR: δ7.90-6.90 (m, 25H, Ph), 5.92 (d, 1H, J=7.5 Hz, NH), 5.53 (dd, 1H,J_(1,2)=1.8 Hz, H-2_(B)), 5.29 (d, 1H, H-1_(E)), 5.19 (m, 2H, H-2_(C),3_(B)), 5.09 (m, 2H, H-1_(C), 1_(D)), 4.97 (bs, 1H, H-1_(B)), 4.96-4.70(m, 9H, CH₂Ph, H-4_(B)), 4.54-4.41 (m, H, CH₂Ph), 4.34 (pt, 1H,J_(3,4)=J_(4,5)=9.3 Hz, H-3_(D)), 4.19-3.89 (m, 6H, H-3_(C), 5_(C),5_(E), 3_(E), 6a_(D), OCH₂), 3.79-3.60 (m, 5H, H-6b_(D), 4_(C), 5_(B),2_(E), OCH₂), 3.56-3.33 (m, 4H, H-5_(D), 4_(E), 4_(D), CH₂N₃), 3.27-3.12(m, 2H, CH₂N₃, H-2_(D)), 2.10, 2.09 (2s, 6H, C(CH₃)₂), 1.78 (s, 3H,OAc), 1.73 (s, 3H, NHAc), 1.42, 1.35 (2s, 6H, OAc), 1.30 (d, 3H,J_(5,6)=6.2 Hz, H-6_(C)), 0.90 (d, 3H, J_(5,6)=6.2 Hz, H-6_(B)); ¹³CNMR: δ 171.4, 169.7, 169.6, 169.5, 166.0 (5C, C═O), 138.7-127.2 (Ph),99.8, 99.7 (C-1_(D), 1_(C)), 97.1 (C-1_(B)), 96.4 (C-1_(E)), 81.5(C-3_(E)), 81.1 (C-2_(E)), 79.5 (bs, C-3_(C)), 77.9 (C-4_(D)), 77.0 (bs,C-4_(C)), 75.4 (C-3_(D)), 75.3, 74.7, 73.6 (3C, CH₂Ph), 73.0, 72.9 (2C,C-2_(C), 4_(E)), 72.9 (CH₂Pb), 71.2 (C-5_(E)), 71.1 (C-4_(B)), 69.9(C-2_(B)), 69.2 (C-6_(E)), 68.8 (C-3_(B)), 68.7 (OCH₂), 67.2, 67.1 (3C,C-5_(C), 5_(B), 5_(D)), 62.2 (C-6_(D)), 59.0 (C-2_(D)), 50.6 (CH₂N₃),29.0, 23.4 (2C, C(CH₃)₂), 20.9, 20.4 (3C, OAc), 19.0 (NHAc), 18.4(C-6_(C)), 17.0 (C-6_(B)). FAB-MS for C₇₂H₈₆N₄O₂₃ (1374) m/z 1397[M+Na]⁺. Anal. Calcd. for C₇₂H₈₆N₄O₂₃: C, 62.87; H, 6.30; N, 4.07%.Found: C, 63.51; H, 6.66; N, 3.77%.

2-Azidoethyl(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(524). 50% aq TFA (1.3 mL) was added to a solution of the fullyprotected tetrasaccharide 523 (210 mg, 111 μmol) in DCM (6 mL). Themixture was stirred at 0° C. for 1 h. Volatiles were evaporated andtoluene was co-evaporated from the residue. Column chromatography(solvent B, 7:3→1:1) of the crude product gave 524 (195 mg, 95%).[α]_(D)−6.9 (c 0.5, MeOH); ¹H NMR: δ 8.08-7.14 (m, 25H, Ph), 5.78 (d,1H, J_(2,NH)=7.4 Hz, NH), 5.51 (br s, 1H, H-2_(B)), 5.27 (d, 1H,J_(1,2)=J_(2,3)=2.9 Hz, H-2_(C)), 5.18 (m, 2H, H-1_(E), 3_(B)), 5.12 (brs, 1H, H-1_(B)), 5.08 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 5.00 (d, 1H,J_(1,2)=2.4 Hz, H-1_(C)), 4.97 (d, 1H, J=11.0 Hz, CH₂Ph), 4.94 (pt, 1H,J_(3,4)=J_(4,5)=9.9 Hz, H-4_(B)), 4.87-4.24 (m, 7H, CH₂Ph), 4.21 (dd,1H, J_(2,3)=8.0, J_(3,4)=10.2 Hz, H-3_(D)), 4.19 (dd, 1H, J_(2,3)=3.2,J_(3,4)=7.9 Hz, H-3_(C)), 4.10-4.04 (m, 2H, H-5_(C), 5_(E)), 4.03 (pt,1H, J_(2,3)=J_(3,4)=9.4 Hz, H-3_(E)), 3.96 (dd, 1H, J_(5,6a)=3.5,J_(6a,6b)=12.5 Hz, H-6a_(D)), 3.85 (dd, 1H, J_(5,6b)=4.0 Hz, H-6b_(D)),3.77-3.70 (m, 5H, H-4_(C), 6a_(E), 6b_(E), OCH₂), 3.68 (m, 1H,J_(4,5)=9.8 Hz, H-5_(B)), 3.63 (dd, 1H, J_(1,2)=3.4, J_(2,3)=9.8 Hz,H-2_(E)), 3.60 (dd, 1H, J_(4,5)=9.6 Hz, H-4_(E)), 3.55-3.44 (m, 3H,H-4_(D), 5_(D), CH₂N₃), 3.29 (m, 1H, CH₂N₃), 3.14 (m, 1H, H-2_(D)),2.13, 2.01, 1.82, 1.80 (4s, 12H, CH₃CO), 1.39 (d, 3H, J_(5,6)=6.2 Hz,H-6_(C)), 0.93 (d, 3H, J_(5,6)=6.1 Hz, H-6_(B)); ¹³C NMR: δ 171.5,170.2, 170.1, 170.0, 166.3 (C═O), 139.2-127.9 (Ph), 99.8 (2C, C-1_(B),1_(D)), 99.5 (C-1_(C)), 98.0 (br s, C-1_(E)), 84.3 (C-3_(D)), 82.0(C-3_(E)), 81.1 (C-2_(E)), 78.8 (br s, C-3_(C)), 78.2 (2C, C-4_(C),4_(E)), 75.9 (CH₂Ph), 75.6 (C-4_(D)), 75.2, 74.2, 73.4 (3C, CH₂Ph), 73.0(C-2_(C)), 71.7 (C-5_(E)), 71.4 (C-5_(D)), 71.3 (C-4_(B)), 70.1(C-2_(B)), 69.4 (C-6_(E)), 69.2, 69.1 (C-3_(B), 5_(C)), 68.9 (OCH₂),67.5 (C-5_(B)), 63.2 (C-6_(D)), 57.7 (C-2_(D)), 51.1 (CH₂N₃), 23.8,21.3, 21.0, 20.9 (4C, CH₃CO), 19.1 (C-6_(C)), 17.4 (C-6_(B)). FAB-MS forC₆₉H₈₂N₄O₂₃ (1334) m/z 1357.5 [M+Na]⁺. Anal. Calcd. for C₆₉H₈₂N₄O₂₃.H₂O:C, 60.43; H, 6.32; N, 4.09%. Found: C, 60.56; 6.22, 3.92%.

2-Aminoethylα-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(525). An ice cold solution of 95% aqueous trifluoroacetic acid (2.4 mL)in CH₂Cl₂ (21.6 mL) was added to the tetrasaccharide 523 (1.93 g, 1.40mmol). The mixture was kept at 0° C. for 5 min., then diluted withtoluene and concentrated. Toluene was co-evaporated from the residue.The residue was dissolved in MeOH (65 mL), and a 1M solution of sodiummethoxide in MeOH (3 mL) was added. The mixture was left to stand at rtfor 18 h, then neutralised with Amberlite IR-120 (H⁺) resin, andfiltered. The filtrate was concentrated, and the residue was purified bycolumn chromatography (solvent B, 9:1) to give 524 (1.38 g, 89%) as acolourless foam. The tetrasaccharide 524 (1.38 g, 1.25 mmol) wasdissolved in a mixture of EtOH (35 mL) and EtOAc (3.5 mL). A 1N solutionof aq HCl (2.5 mL) was added. The mixture was stirred under hydrogen inthe presence of 10% Pd/C (1.5 g) for 72 h, then diluted with water andfiltered. The filtrate was concentrated, then lyophilized. The residuewas dissolved in a solution of 5% aq NaHCO₃ and purified by passingfirst through a column of C₁₈ silica (eluting with water), then througha column of Sephadex G₁₀ (eluting with water) to give, afterlyophilization, 525 (693 mg, 77%). Further RP-HPLC purification of 373mg of the latter gave 351 mg of RP-HPLC pure 525. HPLC (215 nm): Rt 4.78min (Kromasil 5 μm C18 100 Å 4.6×250 mm analytical column, using a 0-20%linear gradient over 20 min of CH₃CN in 0.01 M aq TFA at 1 mL/min flowrate). ¹H NMR (D₂O): δ 5.10 (d, 1H, J_(1,2)=3.7 Hz, H-1_(E)), 4.89 (d,1H, J_(1,2)=1.1 Hz, H-1_(B)), 4.73 (d, 1H, J_(1,2)=1.0 Hz, H-1_(C)),4.50 (d, 1H, J_(1,2)=8.6 Hz, H-1_(D)), 4.08 (m, 1H, H-5_(C)), 3.96 (m,1H, H-2_(B)), 3.91 (m, 2H, H-6a_(D), CH₂O), 3.68-3.88 (m, 12H, H-2_(C),2_(D), 3_(B), 3_(C), 4_(B), 4_(C), 5_(B), 5_(E), 6b_(D), 6a_(E), 6b_(E),CH₂O), 3.59 (pt, 1H, H-3_(E)), 3.52 (pt, 1H, H-3_(D)), 3.33-3.48 (m, 4H,H-2_(E), 4_(D), 4_(E), 5_(D)), 3.01 (m, 2H, CH₂NH₂), 1.99 (s, 3H,CH₃C═O), 1.28 (d, 3H, H-6_(C)), 1.18 (d, 3H, H-6_(B)); ¹³C NMR (D₂O): δ174.8 (C═O), 103.2 (C-1_(B)), 101.4 (C-1_(C)), 100.9 (C-1_(D)), 98.6(C-1_(E)), 81.9 (C-3_(D)), 79.0 (C-4_(B)), 76.6 (C-4_(C)), 76.3(C-2_(E)), 72.9 (C-3_(E)), 72.3 (C-5_(E)), 72.3 (C-4_(D)), 71.8(C-3_(C)), 71.1 (C-2_(C)), 70.5 (C-2_(B), 3_(B)), 69.7 (C-4_(B)), 69.5(C-4_(E)), 69.2 (C-5_(D)), 68.8 (2C, C-5_(B), 5_(C)), 67.9 (CH₂O), 61.0(C-6_(D)), 60.8 (C-6_(E)), 55.5 (C-2_(D)), 40.0 (CH₂NH₂), 22.6 (CH₃C═O),18.0 (C-6_(C)). 17.0 (C-6_(B)). FAB-MS for C₂₈H₅₀N₂O₁₉ (718) m/z 741[M+Na]⁺. HRMS (MALDI) Calcd for C₂₈H₅₀N₂O₁₉Na: 741.2905. Found:741.2939.

Allyl(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside(528). TMSOTf (11 μL, 59 μmol) was added to a solution of the rhamnoside526 (2.26 g, 5.88 mmol) and the trichloroacetimidate 527 (4.23 g, 6.82mmol) in anhydrous Et₂O (60 mL) at −70° C. The reaction mixture wasstirred for 8 h while the cooling bath was slowly coming back to rt.Et₃N (100 μL) was added, and the mixture was stirred at rt for 15 min.Solvents were evaporated, and the crude material was purified by columnchromatography (solvent B, 49:1-9:1), to give 528 as a white foam (4.78g, 96%). ¹H NMR: a 8.17-7.12 (m, 25H, Ph), 5.97-5.85 (m, 3H, H-2_(A),3_(A), CH═), 5.67 (pt, 1H, J_(3,4)=9.6 Hz, H-4_(A)), 5.34-5.19 (m, 3H,H-1_(A), CH₂═), 5.01 (d, 1H, J=9.0 Hz, CH₂Ph), 4.92 (d, 1H, J_(1,2)=1.3Hz, H-1_(B)), 4.82-4.74 (m, 2H, CH₂Ph), 4.71 (d, 1H, J=11.8 Hz, OCH₂),4.31 (dq, 1H, J_(4,5)=9.7 Hz, H-5_(A)), 4.21 (m, 1H, OCH₂), 4.10 (dd,1H, H-2_(B)), 4.02 (m, 1H, OCH₂), 3.97 (dd, 1H, J_(2,3)=3.0, J_(3,4)=9.2Hz, H-3_(B)), 3.82 (dq, 1H, J_(4,5)=9.4 Hz, H-5_(B)), 3.71 (pt, 1H,H-4_(B)), 1.43 (d, 3H, J_(5,6)=6.1 Hz, H-6_(B)), 1.37 (d, 3H,J_(5,6)=6.2 Hz, H-6_(A)); ¹³C NMR: δ 166.3, 165.9, 165.7 (3C, C═O),139.0-127.9 (CH═, Ph), 1117.8 (CH₂═), 99.9 (C-1_(A)), 98.3 (C-1_(B)),80.6 (C-4_(B)), 80.2 (C-3_(B)), 76.5 (C-2_(B)), 76.0, 72.9 (2C, CH₂Ph),72.3 (C-4_(A)), 71.0 (C-2_(A)*), 70.4 (C-3_(A)*), 68.7 (C-5_(B)), 68.1(OCR₂), 67.5 (C-5_(A)), 18.4 (C-6_(B)), 18.1 (C-6_(A)). FAB-MS forC₅₀H₅₀O₁₂ (M, 842.3) m/z 865.1 [M+Na]⁺. Anal. Calcd. for C₅₀H₅₀O₁₂: C,71.24; H, 5.98%. Found C, 71.21; H, 5.99%.

(2,3,4-tri-O-Benzoyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranose(529). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (25 mg) was dissolved in THF (10 mL) and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, until the colour had changed toyellow. The solution was then degassed again in an argon stream. Asolution of 528 (4.71 g, 5.59 mmol) in THF (40 mL) was degassed andadded. The mixture was stirred at rt overnight, then concentrated. Theresidue was taken up in acetone (350 mL) and water (82 mL). Mercuricbromide (3.23 g, 8.96 mmol) and mercuric oxide (2.64 g, 12.3 mmol) wereadded to the mixture, which was protected from light. The suspension wasstirred at rt for 1 h, then concentrated. The residue was taken up inCH₂Cl₂ and washed three times with sat aq KI, then with brine. Theorganic phase was dried and concentrated. The residue was purified bycolumn chromatography (solvent B, 3:1) to give 529 (3.87 g, 84%) as acolourless foam. ¹H NMR: δ 8.15-7.12 (m, 25H, Ph), 5.94-5.88 (m, 3H,H-2_(A), 3_(A), CH═), 5.70 (pt, 1H, J_(3,4)=9.7 Hz, H-4_(A)), 5.31 (dd,1H, J_(1,OH)=3.0 Hz, H-1_(B)), 5.28 (bs, 1H, H-1_(A)), 4.98 (d, 1H,J=11.0 Hz, CH₂Ph), 4.82-4.68 (m, 3H, CH₂Ph), 4.31 (dq, 1H, J_(4,5)=9.8Hz, H-5_(A)), 4.13 (dd, 1H, J_(1,2)=2.1 Hz, H-2_(B)), 4.06-3.99 (m, 2H,H-3_(B), 5_(B)), 3.72 (pt, 1H, J_(3,4)=J_(4,5)=9.4 Hz, H-4_(B)), 2.79(bs, 1H, OH-1_(B)), 1.41 (d, 3H, J_(5,6)=6.2 Hz, H-6_(B)), 1.37 (d, 3H,J_(5,6)=6.3 Hz, H-6_(A)); ¹³C NMR: δ 166.2, 165.9, 165.7 (3C, C═O),138.9-127.9 (Ph), 99.7 (C-1_(A)), 94.2 (C-1_(B)), 80.5 (C-4_(B)), 79.6(C-3_(B)), 77.6 (C-2_(B)), 76.5, 72.5 (2C, CH₂Ph), 72.3 (C-4_(A)), 71.0(C-2_(A)*), 70.4 (C-3_(A)*), 68.8 (C-5_(B)), 67.6 (C-5_(A)), 18.5(C-6_(B)*), 18.1 (C-6_(A)*). FAB-MS for C₄₇H₄₆O₁₂ (M, 802.3) m/z 825.1[M+Na]⁺. Anal. Calcd. for C₄₇H₄₆O₁₂.0.5H₂O: C, 69.53; H, 5.84%. Found C,69.55; H, 5.76%.

(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-(1→2)-3,4-di-O-benzyl-α/β-L-rhamnopyranosyltrichloroacetimidate (530). The hemiacetal 529 (3.77 g, 4.71 mmol) wasdissolved in CH₂Cl₂ (15 mL) and the solution was cooled to 0° C.Trichloroacetonitrile (2.5 mL) was added, then DBU (200 μL). The mixturewas stirred at rt for 2 h. Toluene was added, and co-evaporated twicefrom the residue. The crude material was purified by flashchromatography (solvent B, 4:1+0.1% Et₃N) to give 530 as a white foam(4.29 g, 96%). Some hydrolyzed material 529 (121 mg, 3%) was elutednext. The trichloroacetimidate 530, isolated as an α/β, mixture had ¹HNMR (α anomer): δ 8.62 (s, 1H, NH), 8.20-7.18 (m, 25H, Ph), 6.31 (s, 1H,H-1_(B)), 5.94 (dd, 1H, J_(1,2)=1.6 Hz, H-2_(A)), 5.89 (dd, 1H,J_(2,3)=3.4, J_(3,4)=9.9 Hz, H-3_(A)), 5.71 (pt, 1H, H-4_(A)), 5.27 (bs,1H, H-1_(A)), 5.02 (d, 1H, J=10.8 Hz, CH₂Ph), 4.84 (d, 1H, J=11.9 Hz,CH₂Ph), 4.79 (d, 1H, CH₂Ph), 4.72 (d, 1H, CH₂Ph), 4.36 (dq, 1H,J_(4,5)=9.8 Hz, H-5_(A)), 4.13 (dd, 1H, H-2_(B)), 4.03-3.97 (m, 2H,H-3_(B), 5_(B)), 3.80 (pt, 1H, J_(3,4)=9.5 Hz, H-4_(B)), 1.45 (d, 3H,J_(5,6)=6.1 Hz, H-6_(B)), 1.40 (d, 3H, J_(5,6)=6.2 Hz, H-6_(A)); ¹³C NMR(a anomer): δ 166.2, 165.9, 165.7 (3C, C═O), 160.8 (C═NH), 138.6-128.2(Ph), 99.9 (C-1_(A)), 97.2 (C-1_(B)), 91.4 (CCl₃), 79.9 (C-4_(B)), 79.1(C-3_(B)), 76.2 (CH₂Ph), 74.9 (C-2_(B)), 73.3 (CH₂Ph), 72.1 (C-4_(B)),71.7 (C-5_(B)), 71.0 (C-2_(A)), 70.2 (C-3_(A)), 67.8 (C-5_(A)), 18.4(C-6_(B)), 18.0 (C-6_(A)). Anal. Calcd. for C₄₉H₄₆Cl₃NO₁₂: C, 62.13; H,4.89; N, 1.48%. Found C, 61.81; H, 4.86; N, 1.36%.

Allyl(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(533). (a) The acceptor 519 (465 mg, 0.56 mmol) was dissolved in Et₂O (3mL). The solution was cooled to −60° C. and TMSOTf (65 μL, 0.36 mmol)was added. The donor 530 (690 mg, 0.73 mmol) was dissolved in Et₂O (6mL) and added to the acceptor solution in two portions with an intervalof 30 min. The mixture was stirred at −60° C. to −30° C. over 2 h. Et₃N(100 μL) was added. The mixture was concentrated and the residue waspurified by column chromatography (solvent B, 7:1) to give 533 (501 mg,55%).

(b) A solution of the donor 527 (1.41 g, 2.25 mmol) and the acceptor 532(1.07 g, 1.79 mmol) in anhydrous Et₂O (88 mL) was cooled to −60° C.TMSOTf (63 μL) was added, and the mixture was stirred at −60° C. to −20°C. over 2.5 h. Et₃N was added (100 μL). The mixture was concentrated andthe residue was purified by column chromatography (solvent D, 49:1) togive 533 (2.66 g, 92%); [α]_(D)+74.1 (c 0.5); ¹H NMR: δ 7.06-8.11 (m,50H, Ph), 5.88-6.05 (m, 3H, H-2_(A), 3_(A), CH═), 5.71 (t, 1H, H-4_(A)),5.51 (dd, 1H, H-2_(C)), 5.22-5.41 (m, 3H, H-1_(A), CH₂═), 5.14 (d, 1H,J_(1,2)=0.9 Hz, H-1_(B)), 5.10 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)), 4.97(bs, 1H, H-1_(C)), 4.35-5.00 (m, 14H, H-2_(B), 5_(A), 12×CH₂Ph),3.98-4.19 (m, 5H, H-3_(C), 3_(E), 5_(E), OCH₂), 3.43-3.87 (m, 9H,H-2_(E), 3_(B), 4_(B), 4_(C), 4_(E), 5_(B), 5_(C), 6_(E), 6′_(E)), 1.44(d, 3H, H-6_(A)), 1.40 (d, 3H, H-6_(C)), 1.13 (d, 3H, H-6_(B)); ¹³C NMR:δ 165.9, 165.4, 165.1 (C═O), 127.1-138.7 (CH═, Ph), 117.8 (CH₂═), 101.3(C-1_(B)), 99.6 (C-1_(A)), 97.9 (C-1_(E)), 96.1 (C-1_(C)), 81.9(C-3_(E)), 81.0 (C-2_(E)), 80.1 (C-3_(C)), 79.8 (C-4_(B)), 78.9(C-3_(B)), 77.9 (C-4_(C)), 77.4 (C-4_(E)), 75.9 (C-2_(B)), 75.6, 75.0,74.9, 73.9, 72.9 (CH₂Ph), 72.4 (C-2_(C)), 71.9 (C-4_(A)), 71.2(C-5_(E)), 70.9 (CH₂Ph), 70.7 (C-2_(A)*), 70.0 (C-3_(A)*), 69.2(C-5_(B)), 68.5 (OCH₂), 68.1 (C-6_(E)), 67.6 (C-5_(C)), 67.2 (C-5_(A)),18.8 (C-6_(A)), 18.1 (C-6_(C)), 17.8 (C-6_(B)). FAB-MS for C₉₇H₉₈O₂₂(1614) m/z 1637 [M+Na]⁺. Anal. Calcd. for C₉₇H₉₈O₂₂: C, 72.10; H, 6.11%.Found: C, 71.75; H, 6.27%.

(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-(2-O-benzoyl-α/β-L-rhamnopyranose(534). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (12.5 mg) was dissolved in THF (5 mL) and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the colour to change toyellow. The solution was then degassed again in an argon stream. Asolution of 533 (1.138 g, 0.70 mmol) in THF (15 mL) was degassed andadded. The mixture was stirred at rt overnight. The mixture wasconcentrated. The residue was taken up in acetone (7 mL) and water (0.7mL). Mercuric chloride (285 mg, 1.05 mmol) and mercuric oxide (303 mg,1.4 mmol) were added to the mixture, which was protected from light. Themixture was stirred at rt for 1 h, then concentrated. The residue wastaken up in CH₂Cl₂ and washed three times with sat. aq. KI, then withbrine. The organic phase was dried and concentrated. The residue waspurified by column chromatography (solvent B, 7:3) to give 534 (992 mg,90%) as a colourless foam. ¹H NMR: δ 7.05-8.16 (m, 50H, Ph), 5.88-5.93(m, 2H, H-2_(A), 3_(A)), 5.73 (pt, 1H, H-4_(A)), 5.55 (m, 1H, H-2_(C)),5.37 (bs, 1H, H-1_(A)), 5.28 (bs, 1H, H-1_(C)), 5.14 (bs, 1H, H-1_(B)),5.07 (d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.78-4.99 (m, 6H, CH₂Ph),4.31-4.68 (m, 8H, H-2_(B), 5_(A), CH₂Ph), 4.24 (dd, 1H, H-3_(C)),3.99-4.09 (m, 3H, H-3_(E), 5_(C), 5_(E)), 3.82 (pt, 1H, H-4_(C)),3.57-3.76 (m, 5H, H-3_(B), 4_(E), 5_(B), 6a_(E), 6b_(E)), 3.48 (dd, 1H,H-2_(E)), 3.17 (d, 1H, OH), 1.43 (d, 6H, H-6_(A), 6_(C)), 1.14 (d, 3H,H-6_(B)); ¹³C NMR: δ 166.0, 165.6, 165.2 (4C, C═O), 127.2-138.9 (Ph),101.1 (C-1_(B)), 99.7 (C-1_(A)), 98.1 (C-1_(E)), 91.6 (C-1_(C)), 81.9(C-3_(E)), 81.1 (C-2_(E)), 79.9 (C-4_(B)), 79.4 (C-3_(C)), 78.9(C-3_(B)), 78.3 (C-4_(C)), 77.6 (C-4_(E)), 76.1 (C-2_(B)), 75.8, 75.3,75.1, 74.0, 73.1 (5C, CH₂Ph), 72.7 (C-2_(C)), 72.1 (C-4_(A)), 71.4(C-5_(E)), 71.1 (CH₂Ph), 70.8 (C-2_(A)*), 70.2 (C-3_(A)*), 69.4(C-5_(B)), 68.3 (C-6_(E)), 67.7 (C-5_(C)), 67.3 (C-5_(A)), 19.0(C-6_(A)), 18.2 (C-6_(C)), 17.9 (C-6_(B)). FAB-MS for C₉₄H₉₄O₂₂ (1574)m/z 1597 [M+Na]⁺. Anal. Calcd. for C₉₄H₉₄O₂₂: C, 71.65; H, 6.01%. Found:C, 71.48; H, 6.17%.

(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-(2-O-benzoyl-α/β-L-rhamnopyranosyltrichloroacetimidate (506). The hemiacetal 534 (412 mg, 0.26 mmol) wasdissolved in CH₂Cl₂ (5 mL) and the solution was cooled to 0° C.Trichloroacetonitrile (0.26 mL) was added, then DBU (4 μL). The mixturewas stirred at 0° C. for 1 h. The mixture was concentrated and toluenewas co-evaporated from the residue. The residue was purified by flashchromatography (solvent B, 4:1+0.1% Et₃N) to give 506 (393 mg, 88%). ¹HNMR (α-anomer): δ 8.74 (s, 1H, NH), 7.03-8.10 (m, 50H, Ph), 6.42 (d, 1H,J_(1,2)=2.3 Hz, H-1_(C)), 5.87 (m, 2H, H-2_(A), 3_(A)), 5.67 (m, 2H,H-2_(C), 4_(A)), 5.30 (bs, 1H, H-1_(A)), 5.14 (bs, 1H, H-1_(B)), 5.08(d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.74-4.98 (m, 6H, CH₂Ph), 4.23-4.69(m, 9H, H-2_(B), 3_(C), 5_(A), CH₂Ph), 3.88-4.07 (m, 3H, H-3_(E), 5_(B),5_(E)), 3.57-3.74 (m, 7H, H-2_(E), 4_(B), 4_(C), 4_(E), 5_(C), 6a_(E),6b_(E)), 3.50 (dd, 1H, H-3_(B)), 1.38 (d, 6H, H-6_(A), 6_(B)), 1.07 (d,3H, H-6_(C)); ¹³C NMR (α-anomer): δ 165.9, 165.5, 165.4, 165.1 (4C,C═O), 160.1 (C═NH), 127.2-138.7 (Ph), 101.2 (C-1_(B)), 99.7 (C-1_(A)),98.3 (C-1_(E)), 94.3 (C-1_(C)), 90.9 (CCl₃), 81.7 (C-3_(E)), 80.9(C-2_(E)), 79.6 (C-3_(C), 4_(B)), 78.5 (C-3_(B)), 77.2 (C-4_(C)), 77.5(C-4_(E)), 75.9 (C-2_(B)), 75.6, 75.1, 75.0, 74.0, 72.9 (CH₂Ph), 71.8(C-2_(C)), 71.3 (C-4_(A)), 71.0 (CH₂Ph), 70.7 (C-5_(E)), 70.5(C-2_(A)*), 70.3 (C-3_(A)*), 70.0 (C-5_(B)), 69.5 (C-5_(C)), 67.9(C-6_(E)), 67.2 (C-5_(A)), 18.7 (C-6_(A)), 17.8 (C-6_(C)), 17.7(C-6_(B)). Anal. Calcd. for C₉₆H₉₄Cl₃NO₂₂.H₂O: C, 66.34; H, 5.57; N,0.81%. Found: C, 66.26, H, 5.72; N, 0.94%.

2-Azidoethyl(2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(535). (a) The tetrasaccharide donor 506 (500 mg, 0.29 mmol) and theacceptor 507 (140 mg, 0.42 mmol) were dissolved in 1,2-dichloroethane (5mL) and 4 Å-MS (400 mg) were added. The mixture was stirred at rt for 2h. The mixture was cooled to 0° C. and triflic acid (7 μL, 0.072 mmol)was added. The mixture was stirred at 0° C. to rt over 1 h 30 min. Themixture was then heated at 65° C. for 1 h 30 min. The mixture wasallowed to cool, Et₃N (0.5 mL) was added, and the mixture was stirred atrt for 20 min. The mixture was diluted with CH₂Cl₂ and filtered througha pad of Celite. The filtrate was concentrated and purified by columnchromatography (solvent B, 4:3) to give 535 (340 mg, 62%).

(b) The tetrasaccharide donor 506 (250 mg, 145 μmol) and the acceptor507 (67 mg, 204 μmol) were dissolved in DCM (1.5 mL) and 4 Å-MS (200 mg)were added. The mixture was stirred at −40° C. for 30 min and triflicacid (5 μL) was added. The mixture was stirred at rt over 3 h,triethylamine was added, and the mixture was stirred at rt for 15 min.The mixture was diluted with CH₂Cl₂ and filtered through a pad ofCelite. The filtrate was concentrated and purified by columnchromatography (solvent B, 9:1→1:1) to give 535 (219 mg, 80%).[α]_(D)+64.0 (c 0.1); ¹H NMR: δ 7.04-8.06 (m, 50H, Ph), 6.24 (d, 1H,NH), 5.90 (m, 2H, H-2_(A), 3_(A)), 5.70 (t, 1H, H-4_(A)), 5.42 (m, 1H,H-2_(C)), 5.35 (bs, 1H, H-1_(A)), 5.13 (m, 3H, H-1_(B), 1_(D), 1_(E)),4.77-5.00 (m, 5H, H-1_(C), CH₂Ph), 4.29-4.66 (m, 11H, H-2_(B), 3_(D),5_(A), CH₂Ph), 3.80-4.11 (m, 6H, H-3_(C), 3_(E), 5_(C), 5_(E), 6a_(D),CH₂O), 3.45-3.78 (m, 12H, H-2_(E), 3_(B), 4_(B), 4_(C), 4_(D), 4_(E),5_(B), 5_(D), 6b_(D), 6a_(E), 6b_(E), CH₂O), 3.39 (m, 1H, CH₂N₃), 3.23(m, 2H, H-2_(D), CH₂N₃), 2.13 (s, 3H, CH₃CO), 1.43 (d, 9H, H-6_(A),(CH₃)2C), 1.29 (d, 3H, H-6_(C)), 1.11 (d, 3H, H-6_(B)); ¹³C NMR: δ171.8, 165.9, 165.5, 165.0, 163.5 (5C, C═O), 127.1-138.7 (Ph), 101.3(C-1_(B)), 99.8 (C-1_(D)), 99.3 (C-1_(A)), 97.7 (C-1_(C)), 97.6(C-1_(E)), 91.8 (C(CH₃)₂), 81.6 (C-3_(E)), 81.0 (C-2_(E)), 80.0(C-3_(C)), 79.7 (C-4_(D)), 78.9 (C-4_(B)), 77.5 (C-3_(B), 4_(C)), 76.4(C-3_(D)), 75.6 (C-2_(B)), 75.5, 74.9, 74.8, 73.8, 73.0 (5C, CH₂Ph),72.9 (C-4_(E)), 72.7 (C-2_(C)), 71.8 (C-4_(A)), 71.3 (C-5_(E)), 71.0(CH₂Ph), 70.6 (C-2_(A)*), 70.0 (C-3_(A)*), 69.3 (C-5_(B)), 68.6 (OCH₂),68.3 (C-6_(E)), 67.5 (C-5_(C)), 67.3 (C-5_(A)), 67.1 (C-5_(D)), 62.2(C-6_(D)), 58.9 (C-2_(D)), 50.6 (CH₂N₃), 29.1 (CH₃C), 23.6 (CH₃C═O),19.2 (CH₃C), 18.6 (C-6_(A)), 18.0 (C-6_(C)), 17.6 (C-6_(B)). FAB-MS forC₁₀₇H₁₁₄N₄O₂₇ (1886) m/z 1909 [M+Na]⁺. Anal. Calcd. for C₁₀₇H₁₁₄N₄O₂₇:C, 68.07, H, 6.09; N, 2.97%. Found: C, 68.18, H, 6.07; N, 2.79%.

2-Aminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl)-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(537). An ice cold solution of 95% aq TFA (2.1 mL) in CH₂Cl₂ (8 mL) wasadded to the pentasaccharide 535 (283 mg, 0.15 mmol). The mixture waskept at 0° C. for 2 h, then diluted with toluene and concentrated.Toluene was co-evaporated from the residue. Chromatography of theresidue (solvent B, 7:3→1:1) gave the intermediate diol (265 mg, 96%).The latter (265 mg) was dissolved in MeOH (6 mL), and a 1% solution ofmethanolic sodium methoxide (4.0 mL) was added. The mixture was stirredat 55° C. for 2 h, then neutralised with Dowex X8 (H⁺) resin andfiltered. The filtrate was concentrated. The mixture was purified bycolumn chromatography (solvent A, 100:0→95:5) to give 536 (195 mg, 87%)as a colourless foam, whose structure was confirmed from massspectrometry analysis (FAB-MS for C₇₆H₉₄N₄O₂₃ (M, 1430) m/z 1453[M+Na]⁺). Pentasaccharide 536 (171 mg, 0.11 mmol) was dissolved in EtOH(18 mL). A 1 M solution of aq HCl (210 μL) was added. The mixture wasstirred under hydrogen in the presence of 10% Pd/C (96 mg) for 2 h. Themixture was diluted with EtOH and water, then filtered through a pad ofCelite. The filtrate was concentrated, and preliminary purified bypassing first through a column of C₁₈ silica (eluting with water). Theresidue was purified by RP-HPLC to give, after lyophilization, 537 (50mg, 53%). HPLC (215 nm): Rt 5.87 min (Kromasil 5 μm C18 100 Å 4.6×250 mmanalytical column, using a 0-20% linear gradient over 20 min of CH₃CN in0.01M aq TFA at 1 mL/min flow rate). ¹H NMR (D₂O): δ 5.15 (d, 1H,J_(1,2)=3.7 Hz, H-1_(E)), 5.00 (bs, 1H, H-1_(A)), 4.92 (d, 1H,J_(1,2)=1.1 Hz, H-1_(B)), 4.76 (bs, 1H, H-1_(C)), 4.53 (d, 1H,J_(1,2)=8.6 Hz, H-1_(D)), 4.10 (m, 1H, H-5_(C)), 4.03 (m, 2H, H-2_(A),2_(B)), 4.01 (m, 3H, H-4_(A), 4_(B), CH₂O), 3.83-3.88 (m, 7H, H-2_(C),2_(D), 3_(A), 6a_(D), 6b_(D), 6a_(E), CH₂O), 3.69-3.76 (m, 7H, H-3_(B),3_(C), 3_(E), 4_(C), 5_(A), 5_(B), 6b_(E)), 3.52 (pt, 1H, H-3_(D)),3.33-3.54 (m, 5H, H-2_(E), 4_(D), 4_(E), 5_(D), 5_(E)), 3.09 (m, 2H,CH₂NH₂), 1.98 (s, 3H, CH₃C═O), 1.28 (d, 3H, H-6_(C)), 1.22 (m, 6H,H-6_(A), 6_(B)); ¹³C NMR (D₂O): δ 175.3 (C═O), 103.4 (C-1_(B)), 101.9(C-1_(A)), 101.4 (C-1_(C), 1_(D)), 98.4 (C-1_(E)), 82.3 (C-3_(D)), 80.2(C-2_(B)), 79.9, 76.7 (C-2_(E)), 72.9, 72.4, 72.4, 72.2, 71.8, 71.6,70.5, 70.4, 70.1, 70.0, 69.7, 69.6, 69.4, 68.7, 66.7 (CH₂O), 61.0 (2C,C-6_(D), 6_(E)), 55.5 (C-2_(D)), 39.9 (CH₂NH₂), 22.6 (CH₃C═O), 18.2(C-6_(C)), 17.2 (C-6_(A)), 17.0 (C-6_(B)). HRMS (MALDI) Calcd forC₃₄H₆₀N₂O₂₃+H, 865.3665. Found: 865.3499.

Maleimido Activated PADRE Lys (508).

Starting from 0.1 mmol of Fmoc Pal Peg Ps resin, amino acids (0.4 mmol)were incorporated using HATU/DIEA (0.4 mmol) activation. The N-terminalD-Ala was incorporated as Boc-D-Ala-OH. After completion of the chainelongation, the resin was treated three times with hydrazine monohydrate(2% solution in DMF, 25 mL/g of peptide resin) for 3 min, which allowedthe selective deblocking of the Dde protecting group. To a solution ofmaleimide butyric acid (183 mg, 1.0 mmol) in DCM (2 mL) was added DCC(103 mg, 0.5 mmol). After stirring for 10 min, the suspension wasfiltered, and the filtrate was added to the drained peptide resin. DIEA(17 μL, 0.5 mmol) was added. After 30 min, the peptide resin was washedwith DMF (100 mL), MeOH (100 mL), and dried under vacuum. AfterTFA/TIS/H₂O (95/2.5/2.5) cleavage (10 mL/g of resin, 1.5 h), the crudepeptide (157 mg) was dissolved in 16 mL of 15% CH₃CN in 0.08% aq TFA,and purified by reverse phase Medium Pressure Liquid Chromatography(MPLC) on a Nucleoprep 20 μm C18 100 Å column, using a 15-75% lineargradient of CH₃CN in 0.08% aq TFA over 60 min at 25 mL/min flow rate(214 nm detection) to give 508 (107 mg, 61%). HPLC (214 nm): Rt 13.4 min(94% pure, Nucleosil 5 μm C18 300 Å analytical column, using a 15-45%linear gradient over 20 min of CH₃CN in 0.08% aq TFA at 25 mL/min flowrate). Positive ion ES-MS Calcd for C₈₅H₁₃₉N₂₁O₁₉: 1759.18. Found:1758.83 (SD: 0.40).

(S-Acetylthiomethyl)carbonylaminoethylα-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(538). The trisaccharide 518 (58 mg, 0.1 mmol) was dissolved in DMF (1mL). SAMA-Pfp (33 mg, 0.11 mmol) was added, and the mixture was left tostand at rt for 40 min. Toluene was added and the mixture wasconcentrated. Ether was added to the residue. The resulting precipitatewas collected and purified by passing through a column of C₁₈ silica(water-acetonitrile, gradient) to give 538 (36 mg, 53%). HPLC (230 nm):Rt 13.74 min (99% pure, Kromasil 5 μm C18 100 Å 4.6×250 mm analyticalcolumn, using a 0-20% linear gradient over 20 min of CH₃CN in 0.01M aqTFA at 1 mL/min flow rate). ¹³C NMR (D₂O): δ 200.3 (SC═O), 175.2, 171.9(NC═O), 102.1 (C-1_(C)), 101.2 (C-1_(D)), 100.5 (C-1_(E)), 82.7(C-3_(D)), 81.8 (C-4_(C)), 76.8 (C-2_(E)), 73.6 (C-3_(E)), 72.6(C-5_(E)), 72.4 (C-4_(D)), 71.8 (C-2_(C)), 70.2 (C-4_(E)), 69.7(C-3_(C)), 69.4 (C-5_(D)), 68.9 (C-5_(C)), 68.9 (CH₂O), 61.6 (C-6_(D)),60.9 (C-6_(E)), 56.1 (C-2_(D)), 40.6 (CH₂NH), 33.7 (CH₂S), 30.4(CH₃C(O)S), 23.0 (CH₃C(O)N), 17.5 (C-6_(C)). ES-MS for C₂₆H₄₄N₂O₁₇S(688) m/z 689 [M+H]⁺. HRMS (MALDI) Calcd for C₂₂H₄₄N₂O₁₇SNa: 711.2258.Found: 711.2294.

(S-Acetylthiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(539). A solution of SAMA-Pfp (16.7 mg, 40 μmol) in CH₃CN (150 μL) wasadded to the tetrasaccharide 525 (20 mg, 28.8 μmol) in 0.1 M phosphatebuffer (pH 7.4, 600 μL). The mixture was stirred at rt for 45 min andpurified by RP-HPLC to give 539 (17 mg, 74%). HPLC (230 nm): Rt 13.63min (98% pure, Kromasil 5 μm C18 100 Å 4.6×250 mm analytical column,using a 0-20% linear gradient over 20 min of CH₃CN in 0.01M aq TFA at 1mL/min flow rate). ¹H NMR (D₂O): δ 5.10 (d, 1H, J_(1,2)=3.7 Hz,H-1_(E)), 4.91 (d, 1H, J_(1,2)=0.8 Hz, H-1_(B)), 4.73 (bs, 1H, H-1_(C)),4.45 (d, 1H, J_(1,2)=8.5 Hz, H-1_(D)), 4.09 (m, 1H, H-5_(C)), 3.97 (m,1H, H-2_(B)), 3.87 (m, 4H, H-2_(C), 3_(C), 6a_(D), CH₂O), 3.62-3.78 (m,8H, H-2_(D), 3_(B), 4_(C), 5_(B), 6b_(D), 6a_(E), 6b_(E), 1×CH₂O), 3.60(m, 3H, H-3_(E), CH₂S), 3.48 (pt, 1H, H-3_(D)), 3.39-3.46 (m, 6H,H-2_(E), 4_(B), 4_(D), 4_(E), 5_(D), 5_(E)), 3.33 (m, 2H, CH₂NH₂), 2.35(s, 3H, CH₃C(O)S), 1.98 (s, 3H, CH₃C(O)N), 1.27 (d, 3H, H-6_(C)), 1.23(d, 3H, H-6_(B)): ¹³C NMR (D₂O): δ 199.8 (SC═O), 174.5, 171.3 (NC(O)),103.2 (C-1_(B)), 101.4 (C-1_(C)), 100.9 (C-1_(D)), 98.6 (C-1_(E)), 82.0(C-3_(D)), 79.0 (C-4_(B)), 76.6 (C-4_(C)), 76.3 (C-2_(E)), 72.9(C-3_(E)), 72.3 (C-5_(E)), 72.2 (C-4_(D)), 71.8 (C-3_(C)), 71.0(C-2_(C)), 70.5 (C-2_(B), 3_(B)), 69.7 (C-4_(B)), 69.5 (C-4_(E)), 69.1(C-5_(C), 5_(D)), 68.8 (C-5_(B)), 68.7 (CH₂O), 61.1 (C-6_(D)), 60.7(C-6_(E)), 55.5 (C-2_(D)), 40.1 (CH₂NH), 33.2 (CH₂S), 29.9 (CH₃C(O)S),22.6 (CH₃C(O)N), 17.9 (C-6_(C)), 16.9 (C-6_(B)). MS for C₃₂H₅₄N₂O₂₁S(834) m/z 857 [M+Na]⁺. HRMS-MALDI Calcd for C₃₂H₅₄N₂O₂₁S+Na: 857.2838.Found: 857.2576.

(S-Acetylthiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl)-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(540). The pentasaccharide 537 (6.4 mg, 7.4 μmol) was dissolved in 0.1 Mphosphate buffer (pH 7.4, 1.0 mL). SAMA-Pfp (6.6 mg, 22 μmol) was added,and the mixture was stirred at rt for 5 h. More SAMA-Pfp (6.6 mg, 22μmol) was added and the mixture was stirred for 1 h more at rt. RP-HPLCof the mixture gave 540 (5.4 mg, 75%). HPLC (230 nm): Rt 13.86 min (100%pure, Kromasil-5 μm C18 100 Å 4.6×250 mm analytical column, using a0-20% linear gradient over 20 min of CH₃CN in 0.01M aq TFA at 1 mL/minflow rate). ¹H NMR (D₂O): δ 5.13 (d, 1H, J_(1,2)=3.7 Hz, H-1_(E)), 4.98(bs, 1H, H-1_(A)), 4.90 (bs, 1H, H-1_(B)), 4.74 (bs, 1H, H-1_(C)), 4.47(d, 1H, J_(1,2)=8.5 Hz, H-1_(D)), 4.09 (m, 1H, H-5_(C)), 4.00 (m, 2H,H-2_(A), 2_(B)), 3.79-3.85 (m, 8H, H-2_(C), 2_(D), 3_(A), 4_(A), 4_(B),6a_(D), 6b_(D), CH₂O), 3.65-3.74 (m, 9H, H-3_(B), 3_(C), 3_(E), 4_(C),5_(A), 5_(B), 6a_(E), 6b_(E), CH₂O), 3.60 (m, 2H, CH₂S), 3.53 (pt, 1H,H-3_(D)), 3.13-3.49 (m, 7H, H-2_(E), 4_(D), 4_(E), 5_(D), 5_(E), CH₂NH),2.35 (s, 3H, CH₃C═OS), 1.99 (s, 3H, CH₃C═ON), 1.28 (d, 3H, H-6_(C)),1.20 (m, 6H, H-6_(A), 6_(B)); ¹³C NMR (D₂O): δ 199.9 (SC═O), 174.5,171.4 (NC═O), 102.8 (C-1_(B)), 101.7 (C-1_(A)), 101.4 (C-1_(C)), 100.9(C-1_(D)), 97.9 (C-1_(E)), 82.0 (C-3_(D)), 79.7 (C-2_(B)), 79.0, 76.3,72.9, 72.4, 72.2, 71.8, 71.0, 70.5, 69.7, 69.5, 69.1, 68.8, 68.5 (CH₂O),61.2, 61.0 (2C, C-6_(D), 6_(E)), 55.6 (C-2_(D)), 40.1 (CH₂NH), 33.2(CH₂S), 29.9 (CH₃C═OS), 22.7 (CH₃C═ON), 18.2 (C-6_(C)), 17.2 (C-6_(A)),17.0 (C-6_(B)). HRMS (MALDI) Calcd for C₃₈H₆₄N₂O₂₅SNa: 1003.3417. Found:1003.3426.

PADRE-Lys-(thiomethyl)carbonylaminoethylα-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(501). Compound 538 (5.0 mg, 7.3 μmol) was dissolved in water (500 μL)and added to a solution of 508 (10 mg, 5.68 μmol) in a mixture of water(900 μL), acetonitrile (100 μL) and 0.1M phosphate buffer (pH 6.0, 1mL). 117 μL of a solution of hydroxylamine hydrochloride (139 mg/mL) in0.1M phosphate buffer (pH 6.0) was added and the mixture was stirred for1 h. RP-HPLC purification gave the pure glycopeptide 501 (8.5 mg, 62%).HPLC (230 nm): Rt 10.40 min (100% pure, Kromasil 5 μm C18 100 Å 4.6×250mm analytical column, using a 0-20% linear gradient over 20 min of CH₃CNin 0.01 M aq TFA at 1 mL/min flow rate). ES-MS Calcd forC₁₀₉H₁₈₁N₂₃O₃₅S: 2405.85. Found: 2405.52.

PADRE-Lys-(thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl)-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(502). Compound 539 (4.9 mg, 5.8 μmol) was dissolved in water (500 μL)and added to a solution of 508 (13 mg, 7.4 μmol) in a mixture of water(1 mL), acetonitrile (200 μL) and 0.5 M phosphate buffer (pH 5.7, 1.2mL). 117 μL of a solution of hydroxylamine hydrochloride (139 mg/mL) in0.5M phosphate buffer (pH 5.7) was added, and the mixture was stirredfor 1 h. RP-HPLC purification gave the pure glycopeptide 502 (6.7 mg,48%). HPLC (230 nm): Rt 11.60 min (100% pure, Kromasil 5 μm C18 100 Å4.6×250 mm analytical column, using a 20-50% linear gradient over 20 minof CH₃CN in 0.01M aq TFA at 1 mL/min flow rate). ES-MS Calcd forC₁₂₅H₁₉₁N₂₃O₃₉S: 2552.99. Found: 2551.90.

PADRE-Lys-(thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl)-(1→4)]-(α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(503). Compound 540 (5.59 mg, 5.7 μmol) was dissolved in water (500 μL)and added to a solution of 508 (12.6 mg, 7.2 μmol) in a mixture of water(1 mL), acetonitrile (200 μL), which had been previously diluted with0.5 M phosphate buffer (pH 5.7, 1.2 mL). A solution of hydroxylaminehydrochloride (139 mg/mL) in 0.5M phosphate buffer (pH 5.7, 117 μL) wasadded and the mixture was stirred for 1 h. RP-HPLC purification gave thepure glycopeptide 503 (7.1 mg, 46%). HPLC (230 nm): Rt 10.33 min (100%pure, Kromasil 5 μm C18 100 Å 4.6×250 mm analytical column, using a20-50% linear gradient over 20 min of CH₃CN in 0.01 M aq TFA at 1 mL/minflow rate). ES-MS Calcd for C₁₂₁H₂₀₁N₂₃O₄₃S: 2698.14. Found: 2698.09.

F—Synthesis of Two Linear PADRE-Conjugates Bearing a Deca- or aPentasaccharide B Epitope as Potential Synthetic Vaccine AgainstShigella flexneri Serotype 2a Infection

Allyl(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(611). A mixture of 610 (3.14 g, 1.6 mmol), Bu₃SnH (2.5 mL, 9.3 mmol)and AlBN (240 mg) in dry toluene (40 mL) was stirred for 30 min at rtunder a stream of dry Argon, then was heated for 1 h at 100° C., cooledand concentrated. The residue was eluted from a column of silica gelwith 3:2 petroleum ether-EtOAc to give 611 as a white foam (2.0 g, 68%);[α]_(D)+3° (c 1, CHCl₃). ¹H NMR (CDCl₃): S8.00-7.00 (m, 45H, Ph), 5.82(m, 1H, All), 5.58 (d, 1H, J_(2,NH)=8.0 Hz, N—H_(D)), 5.35 (dd, 1H,J_(1,2)=1.0, J_(2,3)=2.3 Hz, H-2_(C)), 5.19 (m, 2H, All), 5.10 (d, 1H,J_(1,2)=1.0 Hz, H-1_(A)), 4.92 (dd, 1H, J_(2,3)=10.5, J_(3,4)=10.5 Hz,H-3_(D)), 4.92 (d, 1H, J_(1,2)=3.3 Hz, H-1_(E)), 4.90 (d, 1H,J_(1,2)=1.7 Hz, H-1_(B)), 4.89 (d, 1H, H-1_(C)), 4.88 (dd, 1H,J_(4,5)=10.0 Hz, H-4_(D)), 4.62 (d, 1H, J_(1,2)=8.5 Hz, H-1_(D)),4.90-4.35 (m, 16H, CH₂Ph), 4.40 (m, 1H, H-2_(B)), 4.10-4.00 (m, 2H,All), 4.08 (dd, 1H, J_(2,3)=2.4 Hz, H-2_(A)), 4.02 (dd, 1H, H-3_(C)),3.91 (m, 1H, H-2_(D)), 3.90-3.70 (m, 11H, H-4_(C), 5_(C), 3_(A), 5_(A),6a_(D), 6b_(D), 3_(E), 4_(E), 5_(E), 6a_(E), 6b_(E)), 3.61 (dd, 1H,J_(3,4)=9.5 Hz, H-3_(B)), 3.55 (m, 1H, H-5_(B)), 3.41-3.40 (m, 3H,H-4_(A), 5_(D), 2_(E)), 3.47 (m, 1H, J_(4,5)=9.5, J_(5,6)=6.1 Hz,H-5_(B)), 3.35-3.33 (m, 3H, H-4_(A), 5_(D), 2_(E)), 3.25 (dd, 1H,H-4_(B)), 1.95, 1.70 (3s, 9H, OAc), 1.65 (s, 3H, NHAc), 1.32 (d, 3H,J_(5,6)=6.1 Hz, H-6_(A)), 1.30 (d, 3H, J_(5,6)=6.0 Hz, H-6_(C)), 0.97(d, 3H, J_(5,6)=6.0 Hz, H-6_(B)). ¹³C NMR: δ 171.1, 170.8, 170.2, 169.6,166.2 (5C, C═O), 138.2-118.5 (Ph, All), 103.1 (C-1_(D)), 101.4(C-1_(B)), 101.2 (C-1_(A)), 98.5 (C-1_(E)), 96.4 (C-1_(C)), 82.2(C-3_(E)), 81.7 (C-2_(E)), 81.7 (C-4_(A)), 80.4 (C-4_(B)), 80.2(C-3_(C)), 79.0 (C-3_(A)), 78.6 (C-3_(B)), 78.1 (C-2_(A)), 77.8(C-4_(C)), 77.6 (C-4_(E)), 76.0, 75.8, 75.4, 74.7, 74.3, 74.2, 73.3,70.5 (8C, CH₂Ph), 74.9 (C-2_(B)), 72.7 (C-2_(C)), 72.6 (C-3_(D)), 71.9(2C, C-5_(E), 5_(D)), 69.1 (C-5_(B)), 68.9 (2C, All, C-5_(A)), 68.3(C-6_(E)), 67.8 (C-5_(C)), 62.3 (C-6_(D)), 54.6 (C-2_(D)), 23.5 (NHAc),21.1, 21.0, 20.8 (3C, OAc), 19.0 (C-6_(C)), 18.4 (C-6_(A)), 18.2(C-6_(B)). FAB-MS of C₁₀₄H₁₁₇NO₂₇ (M, 1913.1), m/z 1936.2 [M+Na]⁺. Anal.Calcd. for C₁₀₄H₁₁₇NO₂₇: C, 68.90; H, 6.50; N, 0.77. Found: C, 68.64; H,6.66; N, 1.05.

Allyl(2-acetamido-4,6-O-isopropylidene-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranoside(613). The pentasaccharide 611 (2.65 g, 1.47 mmol) was dissolved in MeOH(20 mL). MeONa was added until pH 10. The mixture was stirred for 25 minthen treated by IR 120 (H⁺) until neutral pH. The solution was filteredand concentrated. The residue was eluted from a column of silica gelwith 9:1 DCM-MeOH to give the expected triol 612 which was then treatedovernight at rt by 2,2-dimethoxypropane (11 mL, 0.1 mol) and PTSA (20mg, 0.17 mmol) in DMF (20 mL). Et₃N was added and the solutionevaporated. The residue was eluted from a column of silica gel with 1:1cyclohexane-EtOAc and 0.2% of Et₃N to give 613 as a white foam (2.05 g,81% from 611); [α]_(D)+3° (c 1, CHCl₃). ¹H NMR: δ 6.98-8.00 (m, 45H,Ph), 6.17 (bs, 1H, NH_(D)), 5.82 (m, 1H, All), 5.30 (dd, 1H,J_(1,2)=10.0, J_(2,3)=3.0 Hz, H-2_(C)), 5.11-5.25 (m, 2H, All), 5.06(bs, 1H, H-1_(A)), 4.92 (d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.88 (d, 1H,J_(1,2)=1.6 Hz, H-1_(B)), 4.84 (bs, 1H, H-1_(C)), 4.35 (d, 1H, H-1_(D)),4.34 (dd, 1H, H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph), 4.05 (dd, 1H,H-2_(A)), 3.36 (dd, 1H, H-2_(E)), 2.90-4.10 (m, 22H, All, H-2_(D),3_(A), 3_(B), 3_(C), 3_(D), 3_(E), 4_(A), 4_(B), 4_(C), 4_(D), 4_(E),5_(A), 5_(B), 5_(C), 5_(D), 5_(E), 6a_(D), 6b_(D), 6a_(E), 6b_(E)), 1.5(s, 3H, NHAc), 1.2-0.9 (m, 15H, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³CNMR: δ 172.7, 164.9 (2C, C═O), 137.7-116.7 (Ph, All), 102.3 (C-1_(D)),100.2 (C-1_(B)), 100.0 (C-1_(A)), 98.9 (C(CH₃)₂), 97.2 (C-1_(E)), 95.1(C-1_(C)), 82.1, 82.0, 81.8, 81.6, 80.6, 80.3, 79.0, 78.8, 78.3, 77.8,77.6, 75.7, 75.6, 75.0, 74.3, 72.8, 71.8, 71.6, 70.8, 70.3, 69.0, 68.5,67.8, 67.4, 61.9, 60.8, 60.5, 29.4 (C(CH₃)₂), 22.7 (NHAc), 19.0(C(CH₃)₂), 18.9, 18.4, 18.2 (3C, C-6_(A), 6_(B), 6_(C)). FAB-MS forC₁₀₁H₁₁₅NO₂₄ (M, 1726.9) m/z 1749.7 [M+Na]⁺. Anal. Calcd. forC₁₀₁H₁₁₅NO₂₄.H₂O: C, 69.52; H, 6.76; N, 0.80. Found: C, 69.59; H, 6.71;N, 0.57.

Allyl(2-acetamido-3-O-acetyl-4,6-O-isopropylidene-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-]-2-O-benzoyl-α-L-rhamnopyranoside(614). A mixture of 613 (2.05 g, 1.19 mmol) in pyridine (60 mL) wascooled to 0° C. Acetic anhydride (20 mL) was added and the solution wasstirred 2.5 h. The solution was concentrated and coevaporated withtoluene. The residue was eluted from a column of silica gel with 2:1Cyclohexane-EtOAc and 0.2% of Et₃N to give 614 as a white foam (1.99 g,94%); [α]_(D)+1° (c 1, CHCl₃). ¹H NMR: δ 6.95-8.00 (m, 45H, Ph), 5.82(m, 1H, All), 5.46 (d, 1H, J_(2,NH)=8.0 Hz, NH_(D)), 5.29 (dd, 1H,J_(1,2)=1.0, J_(2,3)=3.0 Hz, H-2_(C)), 5.11-5.25 (m, 2H, All), 5.00 (bs,1H, H-1_(A)), 4.90 (d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.85 (d, 1H,J_(1,2)=1.6 Hz, H-1_(B)), 4.83 (bs, 1H, H-1_(C)), 4.70 (dd, 1H,J_(2,3)=J_(3,4)=10.0 Hz, H-3_(D)), 4.44 (d, 1H, H-1_(D)), 4.34 (dd, 1H,H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph), 4.02 (dd, 1H, H-2_(A)), 3.37 (dd,1H, H-2_(E)), 2.90-4.10 (m, 21H, All, H-2_(D), 3_(A), 3_(B), 3_(C),3_(E), 4_(A), 4_(B), 4_(C), 4_(D), 4_(E), 5_(A), 5_(B), 5_(C), 5_(D),5_(E), 6a_(D), 6b_(D), 6a_(E), 6b_(E)), 1.92 (s, 3H, OAc), 1.57 (s, 3H,NHAc), 1.27-0.90 (m, 15H, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³C NMR: δ171.3, 170.3, 166.2 (3C, C═O), 138.7-117.9 (Ph, All), 103.9 (C-1_(D)),101.5 (C-1_(B)), 101.4 (C-1_(A)), 99.9 (C(CH₃)₂), 98.5 (C-1_(E)), 96.3(C-1_(C)), 82.1, 81.7, 81.6, 80.3, 80.1, 78.8, 78.1, 77.8, 76.0, 75.8,75.3, 75.1, 74.7, 74.2, 73.6, 73.3, 72.7, 71.9, 71.4, 70.8, 69.0, 68.8,68.7, 68.4, 68.1, 67.8, 62.1, 55.0 (C-2_(D)), 30.0 (C(CH₃)₂), 23.5(NHAc), 21.6 (OAc), 19.2 (C(CH₃)₂), 19.0, 18.3, 18.2 (3C, C-6_(A),6_(B), 6_(C)). FAB-MS for C₁₀₃H₁₁₇NO₂₅ (M, 1769.0) m/z 1791.9 [M+Na]⁺.Anal. Calcd. for C₁₀₃H₁₁₇NO₂₅: C, 69.93; H, 6.67; N, 0.79. Found: C,69.77; H, 6.84; N, 0.72.

(2-Acetamido-3-O-acetyl-4,6-O-isopropylidene-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-]-2-O-benzoyl-α-L-rhamnopyranosyltrichloroacetimidate (607).1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (50 mg, 58 μmol) was dissolved THF (10 mL), and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the color to change toyellow. The solution was then degassed again in an argon stream. Asolution of 614 (1.8 g, 1.02 mmol) in THF (20 mL) was degassed andadded. The mixture was stirred at rt overnight, then concentrated todryness. The residue was dissolved in acetone (9 mL), then water (2 mL),mercuric chloride (236 mg) and mercuric oxide (200 mg) were addedsuccessively. The mixture protected from light was stirred at rt for 2h, and acetone was evaporated. The resulting suspension was taken up inDCM, washed twice with 50% aq KI, water and satd aq NaCl, dried andconcentrated. The residue was eluted from a column of silica gel with3:2 Cyclohexane-EtOAc and 0.2% Et₃N to give the corresponding hemiacetal615. Trichloroacetonitrile (2.4 mL) and DBU (72 μL) were added to asolution of the residue in anhydrous DCM (24 mL) at 0° C. After 1 h, themixture was concentrated. The residue was eluted from a column of silicagel with 3:2 cyclohexane-EtOAc and 0.2% Et₃N to give 607 as a colourlessoil (1.58 g, 82% from 614); [α]_(D)+2° (c 1, CHCl₃). ¹H NMR: δ 8.62 (s,1H, NH), 6.95-8.00 (m, 45H, Ph), 6.24 (d, 1H, J_(1,2)=2.6 Hz, H-1_(C)),5.48 (dd, 1H, J_(2,3)=3.0 Hz, H-2_(C)), 5.41 (d, 1H, J_(2,NH)=8.4 Hz,NH_(D)), 4.99 (bs, 1H, H-1_(A)), 4.92 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)),4.88 (d, 1H, J_(1,2)=1.6 Hz, H-1_(B)), 4.69 (dd, 1H,J_(2,3)=J_(3,4)=10.0 Hz, H-3_(D)), 4.44 (d, 1H, H-1_(D)), 4.34 (dd, 1H,H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph), 4.02 (dd, 1H, H-2_(A)), 3.38 (dd,1H, H-2_(E)), 2.90-4.10 (m, 19H, H-2_(D), 3_(A), 3_(B), 3_(C), 3_(E),4_(A), 4_(B), 4_(C), 4_(D), 4_(E), 5_(A), 5_(B), 5_(C), 5_(D), 5_(E),6a_(D), 6b_(D), 6a_(E), 6b_(E)), 1.95 (s, 3H, OAc), 1.55 (s, 3H, NHAc),1.30-0.85 (m, 15H, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³C NMR: δ 172.4,171.4, 166.9 (3C, C═O), 140.2-128.9 (Ph), 104.2 (C-1_(D)), 101.4 (2C,C-1_(A), 1_(B)), 101.1 (C(CH₃)₂), 98.0 (C-1_(E)), 94.8 (C-1_(C)), 92.4(CCl₃), 82.1, 81.5, 80.2, 80.1, 78.6, 78.1, 77.8, 77.6, 76.0, 75.8,75.5, 75.0, 74.3, 74.2, 73.5 (C-3_(D)), 73.4, 71.9, 71.4, 71.0, 70.5,69.2, 68.8, 68.3, 68.1, 62.1, 54.9 (C-2_(D)), 29.3 (C(CH₃)₂), 23.4(NHAc), 21.4 (OAc), 19.2 (C(CH₃)₂), 19.0, 18.2, 18.1 (3C, C-6_(A),6_(B), 6_(C)). FAB-MS for C₁₀₂H₁₁₃Cl₃N₂O₂₅ (M, 1873.3) m/z 1896.3[M+Na]⁺. Anal. Calcd. for C₁₀₂H₁₁₃Cl₃N₂O₂₅: C, 65.40; H, 6.08; N, 1.50.Found: C, 65.26; H, 6.02; N, 1.31.

2-Azidoethyl(2-acetamido-3-O-acetyl-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-β-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(616). A mixture of donor 607 (745 mg, 0.4 mmol) and acceptor 608 (170mg, 0.51 mmol), 4 Å molecular sieves and dry 1,2-DCE (12 mL), wasstirred for 1 h then cooled to 0° C. Triflic acid (25 μL) was added. Thestirred mixture was allowed to reach rt in 10 min then stirred again for2.5 h at 75° C. After cooling to rt, Et₃N (100 μL) was added and themixture filtered. After evaporation, the residue was eluted from acolumn of silica gel with 1:2 cyclohexane-EtOAc and 0.2% Et₃N to give616 as a white foam (615 mg, 76%); [α]_(D)+0° (c 1, CHCl₃). ¹H NMR: δ6.95-7.90 (m, 45H, Ph), 6.02 (d, 1H, J_(2,NH)=7.1 Hz, NH_(D)), 5.46 (d,1H, J_(2,NH)=8.6 Hz, NH_(D′)), 5.20 (dd, 1H, J_(1,2)=1.0, J_(2,3)=3.0Hz, H-2_(C)), 5.03 (d, 1H, J_(1,2)=8.1 Hz, H-1_(D)), 5.02 (bs, 1H,H-1_(A)), 4.92 (d, 1H, J_(1,2)=3.1 Hz, H-1_(E)), 4.85 (d, 1H, J_(1,2)1.6Hz, H-1_(B)), 4.82 (bs, 1H, H-1_(C)), 4.70 (dd, 1H, H-3_(D′)), 4.44 (d,1H, H-1_(D′)), 4.30 (dd, 1H, H-2_(B)), 4.20-4.80 (m, 16H, CH₂Ph), 3.99(dd, 1H, H-2_(A)), 3.37 (dd, 1H, H-2_(E)), 2.90-3.95 (m, 29H, H-2_(D),2_(D′), 3_(A), 3_(B), 3_(C), 3_(D), 3_(E), 4_(A), 4_(B), 4_(C), 4_(D),4_(D′), 4_(E), 5_(A), 5_(B), 5_(C), 5_(D), 5_(D′), 5_(E), 6a_(D),6b_(D), 6a_(D′), 6b_(D′), 6a_(E), 6b_(E), OCH₂CH₂N₃), 2.00 (s, 3H,NHAc), 1.92 (s, 3H, OAc), 1.57 (s, 3H, NHAc), 1.27-0.90 (m, 21H, 2C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³C NMR: δ 172.1, 171.5, 170.3, 166.2(4C, C═O), 139.0-127.7 (Ph), 103.9 (C-1_(D′)), 101.7 (C-1_(B)), 101.2(C-1_(A)), 100.0 (C-1_(D)), 99.9, 99.8 (2C, C(CH₃)₂), 98.3 (C-1_(E)),97.8 (C-1_(C)), 82.0, 81.7, 81.5, 80.8, 80.2, 80.1, 78.9, 78.6, 78.0,77.9, 76.0, 75.9, 75.8, 75.3, 74.8, 74.6, 74.2, 74.0, 73.6, 73.5, 73.4,73.0, 71.9, 71.4, 70.8, 69.1, 69.0, 68.8, 68.6, 68.0, 67.7, 67.6, 62.6,62.1, 60.8, 59.7 (C-2_(D)), 55.0 (C-2_(D′)), 51.1 (CH₂N₃), 29.5(C(CH₃)₂), 29.3 (C(CH₃)₂), 23.9 (NHAc), 23.5 (NHAc), 21.3 (OAc), 19.7(C(CH₃)₂), 19.2 (C(CH₃)₂), 18.8, 18.4, 18.2 (3C, C-6_(A), 6_(B), 6_(C)).FAB-MS for C₁₁₃H₁₃₃N₅O₃₀ (M, 2041.3) m/z 2064.2 [M+Na]⁺. Anal. Calcd.for C₁₁₃H₁₃₃N₅O₃₀: C, 66.49; H, 6.57; N, 3.43. Found: C, 65.93; H, 6.57;N, 2.61.

2-Azidoethyl(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(617). The hexasaccharide 616 (615 mg, 0.30 mmol) was dissolved in MeOH(8 mL). MeONa was added until pH 9. The mixture was stirred for 3 h,then treated by IR 120 (H⁺) until neutral pH. The solution was filteredand concentrated. The residue was eluted from a column of silica gelwith 25:1 DCM-MeOH and 0.2% of Et₃N to give 617 as a white foam (590 mg,97%); [α]_(D)+1° (c 1, CHCl₃). ¹H NMR: δ 8.00-7.00 (m, 45H, Ph), 6.10(d, 1H, NH_(D′)), 6.05 (d, 1H, J_(2,NH)=7.4 Hz, NH_(D)), 5.20 (dd, 1H,J_(1,2)=1.7, J_(2,3)=3.0 Hz, H-2_(C)), 5.10 (d, 1H, J_(1,2)=1.0 Hz,H-1_(A)), 4.99 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 4.96 (d, 1H,J_(1,2)=3.2 Hz, H-1_(E)), 4.90 (d, 1H, J_(1,2)=1.0 Hz, H-1_(B)), 4.86(d, 1H, J_(1,2)=1.0 Hz, H-1_(C)), 4.52 (d, 1H, J_(1,2)=7.5 Hz,H-1_(D′)), 4.37 (dd, 1H, H-2_(B)), 4.22 (dd, 1H, H-3_(D)), 4.02 (dd, 1H,H-2_(A)), 4.80-4.00 (m, 16H, CH₂Ph), 4.00-2.95 (m, 30H, H-2_(D), 4_(D),5_(D), 6a_(D), 6b_(D), 2_(E), 3_(E), 4_(E), 5_(E), 6a_(E), 6b_(E),3_(C), 4_(C), 5_(C), 3_(B), 4_(B), 5_(B), 3_(A), 4_(A), 5_(A), 2_(D′),3_(D′), 4_(D′), 5_(D′), 6a_(D′), 6b_(D′), OCH₂CH₂N₃), 2.00-0.92 (6s, 3d,27H, NHAc, C(CH₃)₂, H-6_(A), 6_(B), 6_(C)). ¹³C NMR partial: δ 173.9,172.1, 166.3 (3C, C═O), 140.0-125.0 (Ph), 103.6 (C-1_(D′)), 101.7(C-1_(B)), 101.2 (C-1_(A)), 100.2 (C(CH₃)₂), 100.2 (C-1_(D)), 99.9(C(CH₃)₂), 98.2 (C-1_(E)), 97.8 (C-1_(C)), 51.1 (CH₂N₃), 29.4, 29.3,23.9, 22.8, 19.6, 19.2, 18.9, 18.4, 18.2 (C-6_(A), 6_(B), 6_(C), NHAc,C(CH₃)₂). FAB-MS for C₁₁₁H₁₃₁N₅O₂₉ (M, 1999.2) m/z 2021.8 [M+Na]⁺. Anal.Calcd. for C₁₁₁H₁₃₁N₅O₂₉: C, 66.68; H, 6.60; N, 3.50. Found: C, 66.63;H, 6.78; N, 3.32.

(2-O-Acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-Obenzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-O-benzoyl-α-L-rhamnopyranosyltrichloroacetimidate (606).1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridiumhexafluorophosphate (80 mg, 93 μmol) was dissolved THF (10 mL), and theresulting red solution was degassed in an argon stream. Hydrogen wasthen bubbled through the solution, causing the colour to change toyellow. The solution was then degassed again in an argon stream. Asolution of 609 (2.55 g, 1.67 mmol) in THF (20 mL) was degassed andadded. The mixture was stirred at rt overnight, then concentrated todryness. The residue was dissolved in acetone (15 mL), then water (3mL), mercuric chloride (380 mg) and mercuric oxide (320 mg) were addedsuccessively. The mixture protected from light was stirred at rt for 2h, and acetone was evaporated. The resulting suspension was taken up inDCM, washed twice with 50% aq KI, water and satd aq NaCl, dried andconcentrated. The residue was eluted from a column of silica gel with3:1 petroleum ether-EtOAc to give the corresponding hemiacetal.Trichloroacetonitrile (2.0 mL) and DBU (25 μL) were added to a solutionof the residue in anhydrous DCM (15 mL) at 0° C. After 1 h, the mixturewas concentrated. The residue was eluted from a column of silica gelwith 3:1 petroleum ether-EtOAc and 0.2% Et₃N to give 606 as a white foam(1.5 g, 56%); [α]_(D)+22° (c 1, CHCl₃). ¹H NMR: δ 8.72 (s, 1H, C═NH),8.00-7.00 (m, 45H, Ph), 6.39 (d, 1H, J_(1,2)=2.5 Hz, H-1_(C)), 5.60 (dd,1H, J_(2,3)=3.0 Hz, H-2_(C)), 5.58 (dd, 1H, J_(1,2)=1.7 Hz, J_(2,3)=3.0Hz, H-2_(A)), 5.12 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)), 5.08 (m, 2H,H-1_(A), 1_(B)), 5.00-4.00 (m, 16H, CH₂Ph), 4.20 (dd, 1H, H-3_(C)), 4.05(dd, 1H, H-3_(E)), 4.00-3.35 (m, 14H, H-2_(E), 4_(E), 5_(E), 6a_(E),6b_(E), 4_(C), 5_(C), 2_(B), 3_(B), 4_(B), 5_(B), 3_(A), 4_(A), 5_(A)),2.05 (s, 3H, OAc), 1.42, 1.36 and 1.00 (3d, 9H, H-6_(A), 6_(B), 6_(C)).¹³C NMR: δ 170.3, 165.8 (2C, C═O), 138-127 (Ph), 99.9 (2C, C-1_(A),1_(B)), 98.5 (C-1_(E)), 94.7 (C-1_(C)), 82.1, 81.2, 80.4, 80.0, 79.1,78.1, 78.0, 75.2, 71.7, 71.2, 70.7, 69.5, 69.4, 68.7 (16C, C-2_(A),3_(A), 4_(A), 5_(A), 2_(B), 3_(B), 4_(B), 5_(B), 2_(C), 3_(C), 4_(C),5_(C), 2_(E), 3_(E), 4_(E), 5_(E)), 76.0, 75.7, 75.5, 75.1, 74.3, 73.3,72.2, 71.2 (8C, PhCH₂), 68.5 (C-6_(E)), 21.4 (OAc), 19.2, 18.5, 18.1(C-6_(A), 6_(B), 6_(C)). Anal. Calcd. for C₉₁H₉₆Cl₃NO₂₀: C, 67.05; H,5.94; N, 0.86. Found: C, 66.44; H, 6.21; N, 0.93.

2-Azidoethyl(2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(618). A mixture of alcohol 617 (110 mg, 55 μmol), trichloroacetimidate606 (179 mg, 110 μmol) and 4 Å molecular sieves in anhydrous 1,2-DCE(2.5 mL) was stirred for 1 h under dry Ar. After cooling at −35° C.,triflic acid (5 μL, 50 μmol) was added dropwise and the mixture wasstirred for 2.5 h, while allowed to reach 10° C. Et₃N (25 μL) was added,and the mixture was filtered and concentrated. The residue was elutedfrom a column of silica gel with 4:1 to 3:1 toluene-EtOAc and Et₃N(0.2%) to give 618 as a white foam (158 mg, 82%); [α]_(D)+18° (c 1,CHCl₃). ¹H NMR: δ 8.00-6.90 (90H, m, Ph), 5.90 (d, 1H, J_(2,NH)=7.0 Hz,NH_(D)), 5.58 (d, 1H, J_(2,NH)=7.5 Hz, NH_(D′)), 5.45, 5.22 (m, 2H,J_(1,2)=1.0, J_(2,3)=2.0 Hz, H-2_(C), 2_(C′)), 5.12 (dd, 1H, H-2_(A′)),5.11 (d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 5.05 (d, 1H, J_(1,2)=1.0 Hz,H-1_(A)), 5.01 (d, 1H, J_(1,2)=3.2 Hz, H-1_(E)), 4.96 (d, 1H,J_(1,2)=1.0 Hz, H-1_(C)), 4.94 (m, 2H, H-1_(E), 1_(B)), 4.86 (d, 1H,H-1_(B)), 4.82 (d, 1H, H-1_(C)), 4.72 (d, 1H, H-1_(D′)), 4.70 (d, 1H,H-1_(A′)), 4.90-4.20 (m, 36H, 16 OCH₂Ph, H-2_(B), 2_(B′), 3_(D),3_(D′)), 4.00-2.90 (m, 45H, H-2_(D), 4_(D), 5_(D), 6a_(D), 6b_(D),3_(C), 4_(C), 5_(C), 2_(E), 3_(E), 4_(E), 5_(E), 6a_(E), 6b_(E), 3_(B),4_(B), 5_(B), 2_(A), 3_(A), 4_(A), 5_(A), 2_(D′), 4_(D′), 5_(D′),6a_(D′), 6b_(D′), 3_(C′), 4_(C′), 5_(C′), 2_(E′), 3_(E′), 4_(E′),5_(E′), 6a_(E′), 6b_(E′), 3_(B′), 4_(B′), 5_(B′), 3_(A′), 4_(A′),5_(A′), OCH₂CH₂N₃), 2.00 (s, 3H, NHAc), 1.88 (s, 3H, OAc), 1.86 (s, 3H,NHAc), 1.40-0.82 (m, 30H, H-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′),C(CH₃)₂). ¹³C NMR partial: δ 172.1, 171.4, 170.2, 166.2, 165.9 (5C,C═O), 102.7 (C-1_(D′)), 101.6, 101.2 (2C, C-1_(B), 1_(B′)), 101.1(C-1_(A)), 99.8 (C-1_(D)), 99.7 (C-1_(C)), 98.2 (2C, C-1_(E), 1_(A′)),97.2 (2C, C-1_(C), 1_(E)), 63.3, 62.6 (2C, C-6_(E), 6_(E′)), 60.0, 57.8(2C, C-2_(D), 2_(D′)), 51.0 (CH₂N₃), 29.5, 29.4 (2C, C(CH₃)₂), 24.0 (2C,NHAc), 21.3 (OAc), 19.6, 19.5 (2C, C(CH₃)₂), 19.1, 18.9, 18.8, 18.5,18.2, 18.1 (6C, C-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′)). FAB-MSof C₂₀₀H₂₂₅N₅O₄₈ (M, 3446.9), m/z 3489.5 ([M+Na]⁺).

2-Azidoethyl(2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(619).

To a solution of 618 (630 mg, 181 μmol) in DCM (12 mL) was addeddropwise, at 0° C., a solution of TFA (2 mL) and water (2 mL). Themixture was stirred for 3 h at this temperature, then concentrated bycoevaporation first with water, then with toluene. The residue waseluted from a column of silica gel with 1:1 toluene-EtOAc to give 619 asa white foam (460 mg, 75%); [α]_(D)+9° (c 1, CHCl₃). FAB-MS ofC₁₉₄H₂₁₇N₅O₄₈ (M, 3386.8), m/z 3409.2 ([M+Na]⁺). Anal. Calcd forC₁₉₄H₂₁₇N₅O₄₈.H₂O: C, 68.43; H, 6.45; N, 2.06. Found: C, 68.40; H, 7.02;N, 1.61.

2-Aminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(603). A mixture of 619 (130 mg, 38 μmol) in MeOH (4 mL) was treated byMeONa until pH 9. The mixture was stirred for 1 h at rt, then heated at55° C. overnight. After cooling to rt, IR 120 (H⁺) was added untilneutral pH, and the solution was filtered and concentrated. The residuewas eluted from a column of silica gel with 25:1 to 20:1 DCM-MeOH togive an amorphous residue. A solution of this residue in EtOH (1.5 mL),EtOAc (150 μL), 1M HCl (66 μL, 2 eq) was hydrogenated in the presence ofPd/C (100 mg) for 72 h at rt. The mixture was filtered and concentratedinto a residue which was eluted from a column of C-18 with water,liophilized to afford amorphous 603 as a white foam (41 mg, 71%);[α]_(D)−7° (c 1, water). ¹H NMR (D₂O) partial: δ 4.90 (m, 2H,J_(1,2)=3.5 Hz, H-1_(E), 1_(E′)), 4.82, 4.76, 4.72, 4.67, 4.52, 4.51 (6bs, 6H, H-1_(A), 1_(B), 1_(C), 1_(A′), 1_(B′), 1_(C′)), 4.41 (d, 1H,J_(1,2)=8.6 Hz, H-1_(D)*), 4.29 (d, 1H, J_(1,2)=8.6 Hz, H-1_(D′)*), 1.77(s, 6H, NHAc), 1.15-0.96 (m, 18H, H-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′),6_(C′)); ¹³C NMR partial (D₂O): δ174.8, 174.7 (2C, C═O), 102.6(C-1_(D)*), 102.9, 101.8, 101.6, 101.4, 101.3 (6C, C-1_(A), 1_(B),1_(C), 1_(A′), 1_(B′), 1_(C′)), 100.8 (C-1_(D′)*), 97.9 (2C, C-1_(E),1_(E′)), 56.0, 56.4 (2C, 2 C-6_(D), 6_(D′)), 22.7, 22.6 (2C, NHAc),18.2, 17.2, 17.0, 16.9 (6C, C-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′),6_(C′)). HRMS: calculated for C₆₆H₁₁₃N₅O₄₅+Na: 1690.6544. Found1690.6537.

2-Azidoethyl(2-acetamido-3-O-acetyl-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(621). A mixture of donor 607 (835 mg, 0.44 mmol) and acceptor 617 (590mg, 0.3 mmol), 4 Å molecular sieves and dry 1,2-DCE (12 mL), was stirredfor 1 h, then cooled to −30° C. Triflic acid (35 μL) was added. Thestirred mixture was allowed to reach 5° C. in 2.5 h. Et₃N (150 μL) wasadded, and the mixture was filtered. After evaporation, the residue waseluted from a column of silica gel with 1:2 Cyclohexane-EtOAc and 0.2%Et₃N to give 621 as a white foam (990 mg, 90%); [α]_(D)+10° (c 1,CHCl₃). ¹H NMR (CDCl₃) partial: δ6.95-7.90 (m, 90H, Ph), 5.98 (d, 1H,J_(2,NH)=6.9 Hz, NH_(D)), 5.60 (d, 1H, J_(2,NH)=7.5 Hz, NH_(D)), 5.45(d, 1H, J_(2,NH)=8.5 Hz, NH_(D)), 5.22 (dd, 1H, J_(1,2)=1.0, J_(2,3)=3.0Hz, H-2_(C)), 5.13 (dd, 1H, J_(1,2)=1.0, J_(2,3)=3.0 Hz, H-2_(C)), 5.08(d, 1H, J_(1,2)=8.3 Hz, H-1_(D)), 5.07 (bs, 1H, H-1_(A)), 5.04 (bs, 1H,H-1_(A)), 4.97 (d, 1H, J_(1,2)=3.0 Hz, H-1_(E)), 4.94 (d, 1H,J_(1,2)=3.0 Hz, H-1_(E)), 4.90 (bs, 1H, H-1_(B)), 4.86 (bs, 1H,H-1_(B)), 4.82 (bs, 1H, H-1_(C)), 4.73 (d, 1H, H-1_(D)), 4.70 (bs, 1H,H-1_(C)), 4.43 (d, 1H, H-1_(D)), 4.20-4.80 (m, 16H, CH₂Ph), 2.00, 1.85,1.58 (3s, 9H, NHAc), 1.95 (s, 3H, OAc), 1.37-0.85 (m, 36H, 3 C(CH₃)₂,H-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′)); ¹³C NMR partial: δ171.7, 170.8, 169.8, 165.8, 165.4 (6C, C═O), 139.0-127.7 (Ph), 103.9(C-1_(D)), 102.8 (C-1_(D)), 101.5 (2C, C-1_(B)), 101.3 (C-1_(A)), 101.1(C-1_(A)), 100.0 (C-1_(D)), 99.5, 99.3 (3C, C(CH₃)₂), 98.3 (C-1_(E)),98.1 (2C, C-1_(C), 1_(E)), 97.8 (C-1_(C)), 82.0, 81.7, 81.6, 81.4, 80.3,80.2, 80.1, 79.5, 79.2, 78.9, 78.7, 78.4, 78.1, 77.9, 77.8, 77.6, 76.0,75.8, 75.3, 75.2, 74.7, 74.4, 74.1, 74.0, 73.6, 73.5, 73.4, 73.3, 73.0,72.7, 71.9, 71.4, 70.9, 70.8, 69.1, 69.0, 68.9, 68.7, 68.6, 68.5, 68.1,67.8, 67.7, 67.5, 62.6, 62.3, 62.1, 60.8, 59.9, 57.9, 55.0 (3C, C-2_(D),2_(D′), 2_(D″)), 51.1 (CH₂N₃), 29.5, 29.4, 29.3 (3C, C(CH₃)₂), 24.0,23.9, 23.5 (3C, NHAc), 21.3 (OAc), 19.7, 19.6, 19.2 (3C, C(CH₃)₂), 18.9,18.8, 18.6, 18.5, 18.2, 18.1 (6C, C-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′),6_(C′)). FAB-MS for C₂₁₁H₂₄₂N₆O₅₃ (M, 3710.2) m/z 3733.3 [M+Na]⁺. Anal.Calcd. for C₂₁₁H₂₄₂N₆O₅₃: C, 68.31; H, 6.57; N, 2.27. Found: C, 68.17;H, 6.74; N, 2.12.

2-Azidoethyl(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1-4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(622). The undecasaccharide 621 (990 mg, 0.27 mmol) was dissolved inMeOH (30 mL). MeONa was added until pH 9. The mixture was stirred for 3h, then treated by IR 120 (H⁺) until neutral pH. The solution wasfiltered, and concentrated. The residue was eluted from a column ofsilica gel with 1:1 toluene-EtOAc and 0.2% of Et₃N to give 622 as awhite foam (900 mg, 91%); [α]_(D)+15° (c 1, CHCl₃); ¹H NMR partial:56.95-8.00 (m, 90H, Ph), 6.19 (bs, 1H, NH_(D)*), 5.96 (d, 1H,J_(2,NH)=6.8 Hz, NH_(D′)*), 5.57 (d, 1H, J_(2,NH)=6.8 Hz, NH_(D″)*),5.22 (dd, 1H, H-2_(C)*), 5.13 (dd, 1H, H-2_(C′)*), 5.10 (d, 1H,H-1_(D)), 5.07 (bs, 1H, H-1_(A)*), 5.04 (bs, 1H, H-1_(A′)*), 4.96 (d,1H, H-1_(E)*), 4.94 (d, 1H, H-1_(E′)*), 4.85 (bs, 1H, H-1_(B)*), 4.84(bs, 1H, H-1_(B′)*), 4.82 (bs, 1H, H-1_(C)*), 4.70 (d, 1H, H-1_(C′)*),4.67 (d, 1H, H-1_(D)*), 4.44 (d, 1H, H-1_(D′)*), 4.20-4.80 (m, 16H,CH₂Ph), 2.00, 1.85, 1.58 (3s, 9H, NHAc), 1.37-0.80 (m, 36H, C(CH₃)₂,H-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′)). ¹³C NMR partial: δ172.8, 170.9, 170.3, 165.1, 164.7 (5C, C═O), 139.0-127.7 (Ph), 103.5,103.1 (2C, C-1_(D), 1_(D′)), 101.5 (2C, C-1_(B), 1_(B′)), 101.2, 101.1(2C, C-1_(A), 1_(A′)), 99.9 (C-1_(D″)), 99.0, 98.8, 98.7 (3C, C(CH₃)₂),98.3 (C-1_(E)*), 98.1 (2C, C-1_(C)*, 1_(E′)*), 97.8 (C-1_(C′)*), 82.1,82.0, 81.9, 81.7, 81.6, 81.5, 80.6, 80.3, 80.2, 80.1, 79.7, 79.1, 78.9,78.5, 77.9, 77.6, 75.7, 74.9, 74.6, 74.3, 73.3, 73.0, 72.7, 71.9, 71.8,69.1, 68.9, 68.7, 68.5, 68.0, 67.8, 67.7, 67.6, 67.5, 62.6, 62.3, 61.9,60.5, 59.9, 57.4, 55.0 (3C, C-2_(D), 2_(D′), 2_(D″)), 51.0 (CH₂N₃),29.5, 29.3 (3C, C(CH₃)₂), 24.0, 23.9, 22.7 (3C, NHAc), 19.7, 19.6, 19.3(3C, C(CH₃)₂), 19.0, 18.9, 18.6, 18.5, 18.2, 18.1 (6C, C-6_(A), 6_(B),6_(C), 6_(A′), 6_(B′), 6_(C′)). FAB-MS for C₂₀₉H₂₄₀N₆O₅₂ (M, 3668.1) m/z3690.8 [M+Na]⁺. Anal. Calcd. for C₂₁₁H₂₄₂N₆O₅₃: C, 68.43; H, 6.59; N,2.29. Found: C, 68.28; H, 6.72; N, 2.11.

2-Azidoethyl(2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside(623). A mixture of donor 606 (377 mg, 0.230 mmol) and acceptor 622 (427mg, 0.115 mmol), 4 Å molecular sieves and dry 1,2-DCE (10 mL), wasstirred for 1 h then cooled to −30° C. Triflic acid (20 μL) was added.The stirred mixture was allowed to reach 5° C. in 2.5 h. Et₃N (150 μL)was added, and the mixture filtered. After evaporation, the residue waseluted from a column of silica gel with 3:1 toluene-EtOAc and 0.2% Et₃Nto give 623 as a foam (490 mg, 82%); [α]_(D)+20° (c 1, CHCl₃); ¹H NMRpartial: δ 6.90-8.00 (m, 135H, Ph), 5.95 (d, 1H, J_(2,NH)=6.6 Hz,NH_(D)*), 5.60 (d, 1H, J_(2,NH)=8.0 Hz, NH_(D′)*), 5.59 (d, 1H,J_(2,NH)=7.5 Hz, NH_(D″)*), 5.44 (dd, 1H, H-2_(C)), 5.22 (dd, 1H,H-2_(C)), 5.10 (dd, 1H, H-2_(C)), 2.20 (s, 3H, OAc), 2.00, 1.85, 1.84(3s, 9H, AcNH), 1.40-0.80 (m, 45H, 3 C(CH₃)₂, H-6_(A), 6_(B), 6_(C),6_(A′), 6_(B′), 6_(C′), 6_(A″), 6_(B″), 6_(C″)); ¹³C NMR partial: δ173.2, 172.6, 172.5, 171.3, 167.4, 167.0, 166.9 (C═O), 140.2-126.8 (Ph),102.8, 102.7, 101.5, 101.3, 101.1, 99.9, 99.8, 98.1, 97.8, 82.0, 81.7,81.5, 81.4, 80.2, 80.1, 79.6, 79.4, 78.9, 78.6, 78.0, 77.9, 77.6, 75.5,73.4, 73.3, 73.0, 72.8, 71.9, 71.6, 69.4, 69.1, 69.0, 68.6, 67.8, 67.7,67.6, 67.5, 62.6, 62.3, 60.0, 57.9, 57.7, 51.0 (CH₂N₃), 30.5 (3C,C(CH₃)₂), 25.0, 22.4 (3C, NHAc), 22.9 (OAc), 20.7, 20.6, 20.2 (3C,C(CH₃)₂), 20.0, 19.9, 19.8, 19.7, 19.6, 19.3, 19.2, 19.1 (9C, C-6_(A),6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′), 6_(A″), 6_(B″), 6_(C″)). FAB-MSfor C₂₉₈H₃₃₄N₆O₇₁ (M, 5135.8) m/z 5159.3 [M+Na]⁺. Anal. Calcd. forC₂₉₈H₃₃₄N₆O₇₁: C, 69.69; H, 6.55; N, 1.64. Found: C, 69.74; H, 6.72; N,1.49.

2-Azidoethyl(2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-(2-O-benzoyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(624). To a solution of the pentadecasaccharide 623 (480 mg, 93 μmol) inDCM (14 mL) was added dropwise at 0° C., a solution of 50% aq TFA (3.0mL). The mixture was stirred for 3 h then concentrated by coevaporationfirst with water, then with toluene. The residue was eluted from acolumn of silica gel with 1:1 toluene-EtOAc to give 624 as a white foam(390 mg, 83%); [α]_(D)+12° (c 1, CHCl₃); FAB-MS for C₂₈₉H₃₂₂N₆O₇₁ (M,5015.6) m/z 5037.2 [M+Na]⁺.

2-Aminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(604). A solution of the partially deprotected pentadecasaccharide 624(390 mg, 77 μmol) in MeOH (10 mL) was treated by MeONa until pH 10. Themixture was stirred overnight at 55° C. After cooling at rt, IR 120 (H⁺)was added until neutral pH. The solution was filtered, concentrated, andthe residue was eluted from a column of silica gel with 20:1 DCM-MeOH togive the benzylated residue (252 mg). A solution of this residue in EtOH(3 mL), EtOAc (250 μL) and 1M HCl (106 μL) was hydrogenated in thepresence of Pd/C (300 mg) for 48 h at rt. The mixture was filtered andconcentrated, and the residue was eluted from a column of C-18 withwater/CH₃CN, and freeze-dried to afford amorphous 604 (127 mg, 65%);[α]_(D)−5° (C₁, water). ¹H NMR (D₂O) partial: δ 5.13 (m, 3H, H-1_(E),1_(E′), 1_(E″)), 5.07, 4.99, 4.95, 4.90, 4.75 (m, 9H, H-1_(A), 1_(B),1_(C), 1_(A′), 1_(B′), 1_(C′), 1_(A″), 1_(B″), 1_(C″)), 4.63, 4.51 (2d,3H, J_(1,2)=8.5 Hz, H-1_(D), 1_(D′), 1_(D″)), 2.00 (s, 9H, NHAc),1.30-1.18 (m, 27H, H-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′),6_(A″), 6_(B″), 6_(C″)); ¹³C NMR (D₂O) partial: δ 174.8, 174.7 (3C,C═O), 102.9, 102.6, 101.7, 101.3, 100.8, 97.9, 81.8, 81.7, 79.6, 79.0,76.3, 76.2, 73.0, 72.7, 72.4, 72.1, 71.6, 70.5, 70.1, 70.0, 69.7, 69.6,69.4, 68.7, 68.6, 66.0, 61.0, 56.0, 55.4, 39.8, 22.7, 22.6 (NHAc), 18.2,17.2, 17.0, 16.9 (9C, C-6_(A), 6_(B), 6_(C), 6_(A′), 6_(B′), 6_(C′),6_(A″), 6_(B″), 6_(C″)). MALDI-MS for C₉₈H₁₆₆N₄O₆₇Na (M, 2493.96) m/z2494.96.

(S-Acetylthiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(620). A solution of SAMA-PfP (2.8 mg, 9.5 μmol) in CH₃CN (60 μL) wasadded to the aminoethyl decasaccharide 603 (6.4 mg, 3.84 μmol) in 0.1Mphosphate buffer (pH 7.4, 500 μL). The mixture was stirred at rt for 1 hand purified by RP-HPLC to give 620 (4.2 mg, 61%). HPLC (230 nm): Rt14.17 min (99.9% pure, Kromasil 5 μm C18 100 Å 4.6×250 mm analyticalcolumn, using a 0-20% linear gradient over 20 min of CH₃CN in 0.01 M aqTFA at 1 mL/min flow rate). ES-MS for C₇₀H₁₁₇N₃O₄₇S (M, 1784.76) m/z1784.70.

(S-Acetylthiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(625). A solution of SAMA-Pfp (2.8 mg, 9.6 μmol) in CH₃CN (50 μL) wasadded to the pentadecasaccharide 604 (9.4 mg, 3.8 μmol) in 0.1Mphosphate buffer (pH 7.4, 500 μL). The mixture was stirred at rt for 2 hand purified by RP-HPLC to give 625 (6.3 mg, 63%). HPLC (230 nm): Rt13.97 min (99.0% pure, Kromasil 5 μm C18 100 Å 4.6×250 mm analyticalcolumn, using a 0-20% linear gradient over 20 min of CH₃CN in 0.01M aqTFA at 1 mL/min flow rate. ES-MS for C₁₀₂H₁₇₀N₄O₆₉S (M, 2588.53) m/z2588.67.

PADRE (thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(601). Compound 620 (6.0 mg, 3.36 μmol) was dissolved in water (300 μL)and added to a solution of PADRE-Mal (7.1 mg, 4.0 μmol) in a mixture ofwater (630 μL), CH₃CN (120 μL) and 0.1M phosphate buffer (pH 5.6, 750μL). 68 μL of a solution of hydroxylamine hydrochloride (139 mg/mL) in0.1M phosphate buffer (pH 5.6) was added and the mixture was stirred for2 h. RP-HPLC purification gave the pure target 601 (5.2 mg, 44%). HPLC(230 nm): Rt 10.03 min (100% pure, Kromasil 5 μm C18 100 Å 4.6×250 mmanalytical column, using a 20-50% linear gradient over 20 min of CH₃CNin 0.01M aq TFA at 1 mL/min flow rate). ES-MS Calcd for C₁₅₃H₂₅₄N₂₄O₆₅S(M, 3501.91) m/z 3501.15.

PADRE (thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(602). Compound 625 (10.3 mg, 3.98 μmol) was dissolved in water (350 μL)and added to a solution of PADRE-Mal (9.0 mg, 5.0 μmol) in a mixture ofwater (740 μL), CH₃CN (140 μL) and 0.5M phosphate buffer (pH 5.6, 890μL). 80 μL of a solution of hydroxylamine hydrochloride (139 mg/mL) in0.5M phosphate buffer (pH 5.7) was added, and the mixture was stirredfor 3 h. RP-HPLC purification gave the pure conjugate 602 (11.5 mg,67%). HPLC (230 nm): Rt 9.07 min (100% pure, Kromasil 5 μm C18 100 Å4.6×250 mm analytical column, using a 20-560% linear gradient over 20min of CH₃CN in 0.01M aq TEA at 1 mL/min flow rate). ES-MS Calcd forC₁₈₅H₃₀₇N₂₅O₈₇S (M, 4305.69) m/z 4305.45.

G. Synthesis of Biotinylated Analogues of OligosaccharidesRepresentative of Fragments of the O—SP of Shigella flexneri 2a

(+)-Biotinyl-3,6-dioxaoctainediaminyl-(thiomethyl)carbonylaminoethylα-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(708). Compound 701 (5.0 mg, 7.26 μmol) was dissolved in water (280 μL)and added to a solution of 707 (3.2 mg, 7.26 μmol) in 0.5 M phosphatebuffer (pH 6.0, 400 μL). A 2 M solution of hydroxylamine in 0.5 Mphosphate buffer (150 μL) was added and the mixture was stirred at rtfor 1 h. More 707 (1.5 mg, 2.85 μmol) in 0.5 M phosphate buffer (300 μL)was added, and the mixture was stirred for 1 h30 at rt. RP-HPLCpurification gave the pure neoglycopeptide 708 (5.7 mg, 67%). ES-MS forC₄₇H₇₇N₇O₂₃S₂ (M, 1171.5) m/z 1171.45.

(+)-Biotinyl-3,6-dioxaoctainediaminyl-(thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(709). Compound 702 (10.0 mg, 12.0 μmol) was dissolved in water (500 μL)and added to a solution of 707 (12.6 mg, 20.0 μmol) in 0.5 M phosphatebuffer (pH 6, 220 μL). A 2 M solution of hydroxylamine in 0.5 Mphosphate buffer (300 μL) was added and the mixture was stirred at rtfor 2 h. Since HPLC control showed that some 702 remained, the pH of themixture was adjusted to 5 by dropwise addition of diluted aq NH₃, andthe mixture was stirred for 1 h more at rt. RP-HPLC purification gavethe pure neoglycopeptide 709 (12.6 mg, 80%). ES-MS Calcd forC₁₀₉H₁₈₁N₂₃O₃₅S₂ (M, 2405.85) m/z 1317.51.

(+)-Biotinyl-3,6-dioxaoctainediaminyl-(thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(710). Compound 703 (3.8 mg, 3.87 μmol) was dissolved in water (250 μL)and added to a solution of 707 (3 mg, 5.7 μmol) in 0.5 M phosphatebuffer (pH 5.8, 250 μL). A 2 M solution of hydroxylamine in 0.5 Mphosphate buffer (75 μL) was added and the mixture was stirred at rt for1 h. RP-HPLC purification gave the pure neoglycopeptide 710 (4.6 mg,81%). ES-MS Calcd for C₅₉H₉₇N₇O₃₁S₂ (M, 1464.6) m/z 1463.57.

(+)-Biotinyl-3,6-dioxaoctainediaminyl-(thiomethyl)carbonylaminoethyl2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1″2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(711). Compound 704 (2.5 mg, 2.11 μmol) was dissolved in water (85 μL)and added to a solution of 707 (1.7 mg, 3.2 μmol) in 0.5 M phosphatebuffer (pH 5.9, 215 μL). A 2 M solution of hydroxylamine in 0.5 Mphosphate buffer (45 μL) was added and the mixture was stirred at rt for2 h. RP-HPLC purification gave the pure neoglycopeptide 711 (2.5 mg,71%). HPLC (230 nm): Rt 17.03 min (100% pure, Kromasil 5 μm C18 100 Å4.6×250 mm analytical column, using a 0-30% linear gradient over 20 minof CH₃CN in 0.01M aq TFA at 1 mL/min flow rate). ES-MS forC₆₇H₁₁₀N₈O₃₆S₂ (M, 1667.78) m/z 1667.45.

(+)-Biotinyl-3,6-dioxaoctainediaminyl-(thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(712). Compound 705 (4.0 mg, 2.24 μmol) was dissolved in water (85 μL)and added to a solution of 707 (1.8 mg, 3.3 μmol) in 0.5 M phosphatebuffer (pH 5.9, 220 μL). A 2 M solution of hydroxylamine in 0.5 Mphosphate buffer (45 μL) was added and the mixture was stirred at rt for2 h. RP-HPLC purification gave the pure neoglycopeptide 712 (4.5 mg,89%). HPLC (230 nm): Rt 16.69 min (100% pure, Kromasil 5 μm C18 100 Å4.6×250 mm analytical column, using a 0-30% linear gradient over 20 minof CH₃CN in 0.01 M aq TFA at 1 mL/min flow rate). ES-MS forC₉₁H₁₁₅N₈O₅₃S₂ (M, 2268.35) m/z 2267.72.

(+)-Biotinyl-3,6-dioxaoctainediaminyl-(thiomethyl)carbonylaminoethylα-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside(713). Compound 706 (5.7 mg, 2.21 μmol) was dissolved in water (85 μL)and added to a solution of 707 (1.7 mg, 3.2 μmol) in 0.5 M phosphatebuffer (pH 5.9, 220 μL). A 2 M solution of hydroxylamine in 0.5 Mphosphate buffer (45 μL) was added and the mixture was stirred at rt for2 h. RP-HPLC purification gave the pure neoglycopeptide 713 (4.8 mg,71%). HPLC (230 nm): Rt 16.35 min (100% pure, Kromasil 5 μm C18 100 Å4.6×250 mm analytical column, using a 0-30% linear gradient over 20 minof CH₃CN in 0.01 M aq TFA at 1 mL/min flow rate). ES-MS forC₁₂₃H₂₀₃N₉O₇₅S₂ (M, 3072.13) m/z 3072.17.

II The Serum Immunoglobulin G-Mediated Response to Serotype-SpecificDeterminants of Shigella flexneri Lipopolysaccharide Protects AgainstExperimental Shigellosis

Both intestinal secretory IgA (SIgA) and serum IgG specific for theO-antigen (O—SP, FIG. 29), the polysaccharide part of the bacteriallipopolysaccharide (LPS) are elicited upon Shigella infection, thecausative agent of bacillary dysentery. However, the respectiveprotective roles of local and systemic humoral immunity remain unclear

The ineffectiveness of parenterally injected inactivated whole-cellvaccines in inducing protection, despite the high level of anti-LPSserum IgG antibodies raised, has led to the belief that serum antibodiesdo not confer protection (Formal et al., ProC; Soc. Exp. Biol. Med.,1967, 125, 347-; Higgins et al., Am. J. Trop. Med., 1955, 4, 281-288).However, several indirect pieces of evidence suggest that anti-O—SPserum IgG may confer protection during natural infection. A correlationwas found between the level of anti-LPS IgG antibodies and resistance toshigellosis among Israeli soldiers (Cohen et al., J. Inf. Dis., 1988,157, 1068; Cohe, et al., J. Clin. Microbial., 1991, 29, 386), and aninverse relationship exists between the age of incidence of shigellosisand the presence of IgG antibodies to Shigella LPS (Passwell et al.,Pediatr. Infect. Dis., 1995, 14, 859-; Van de Verg et al., J. Infect.Dis., 1992, 166, 158-161). In addition, a detoxified LPS-based conjugatevaccine administered parenterally and eliciting mainly, if not only,serum antibodies has been shown to induce protective immunity (Cohen etal., lancet, 1997, 349, 155-).

In the current study, using the mouse model of pulmonary infection andspecific polyclonal serum or monoclonal IgG, the protective role ofserum IgG recognizing serotype-specific LPS determinants or peptideepitopes on the invasins IpaB and IpaC was addressed.

A) Materials and Methods

1) Bacterial Strains

M90T, an invasive isolate of S. flexneri serotype 5a, and 454, aninvasive isolate of S. flexneri serotype 2a, were the virulent strainsof reference. For i.n. infection, bacteria were routinely grown on LuriaBertoni agar plates at 37° C. They were recovered from plates andbacterial dilutions were performed in 0.9% NaCl with the considerationthat, for an optical density of 1 at 600 nm, the bacterial concentrationwas 5×10⁸ colony forming units (c.f.u)/ml. Killed bacteria for systemicimmunizations were prepared from bacterial cultures at stationary phase,diluted to 5×10⁸ c.f.u./ml in 0.9% NaCl, and then incubated at 100° C.for 1 h. They were then kept at −20° C. in aliquots.

2) Production and Characterization of mAbs Specific for S. flexneriSerotype 2a and 5a LPS

BALB/c mice were immunized intraperitoneally (i.p.) with 10⁷ c.f.u. ofkilled S. flexneri 5a or S. flexneri 2a bacteria three times at 3week-intervals. Mice eliciting the highest anti-LPS antibody responsewere given an intravenous booster injection 3 days before beingsacrificed for splenic B cell fusion according to Kohler and Milstein(Eur. J. Immunol., 1976, 6, 511-519). Hybridoma culture supernatantswere screened for antibody production by ELISA using LPS purified fromS. flexneri X, Y, 5a, 5b, 2a, 2b, 1a and 3a, respectively. The hybridomacells secreting murine IgG (mIgG) reacting specifically with LPShomologous to the strain used for immunization, i.e. recognizingserotype-specific determinants on the LPS O—SP, were selected. A panelof mIgG representative of the four murine IgG subclasses was used forthe study. Those selected were then cloned by limiting dilution, andinjected i.p. into histocompatible mice for ascitis production. mIgGwere precipitated with 50% ammonium sulfate from ascitis fluid,centrifuged, and dialysed against PBS before being purified usingion-exchange chromatography as previously described (Barzu et al.,Infect. Immun., 1998, 65, 1599-1605; Phalipon et al., Infect. Immun.,1992, 60, 1919-1926). The avidity of anti-LPS mIgG for LPS wasdetermined as follows: various concentrations of LPS were incubated insolution overnight at 4° C. with a defined amount of a given mIgG untilequilibrium was reached. Each mixture was then transferred to amicrotiter plate previously coated with homologous purified LPS. Boundantibodies were detected by using peroxidase-conjugated anti-mouseimmunoglobulins specific for IgG subclasses. IC₅₀ was defined as theconcentration of LPS required to inhibit 50% of mIgG binding to LPS.

3) ELISA

Hybridoma culture supernatants were tested by ELISA for the presence ofanti-LPS antibodies as previously described (Barzu et al., Infect.Immun., 1993, 61, 3825-3831; Phalipon et al., Infect. Immun., 1992, 60,1919-1926) except that LPS purified according to Westphal (MethodsCarbohydr. Chem., 1965, 5, 83-91) was used at a concentration of 5 μg/mlin PBS. As secondary antibodies, anti-mouse IgG- or IgM- or IgA-alkalinephosphatase-labeled conjugate (SIGMA) were used at a dilution of1:5,000. To measure the anti-LPS antibody titer in polyclonal serum,biotin-labeled Abs to IgG and its different subclasses (IgG1, -2a, -2b,-3) (PHARMINGEN) and avidin conjugated with alkaline phosphatase (SIGMA)were used at a dilution of 1:5,000. Antibody titers were defined as thelast dilution of the sample giving an OD at least twice that of thecontrol.

4) Active and Passive Immunization of Mice

To obtain polyclonal serum, mice were immunized i.p. with 5×10⁷ killedbacteria, three times at 3 week-intervals. After bleeding, anti-LPSantibody titer in the polyclonal sera was measured by ELISA, asdescribed above, and those ranging from low (1/4,000) to high titer(1/64,000) were used for i.n. passive transfer. Purified mAbs (20 or 2μg) were also administered intranasally. All i.n. administrations wereperformed using a volume of 20 μl and mice previously anesthesized viathe intramuscular route with 50 μl of a mixture of 12.5% ketamine(MERIAL) and 12.5% acepromazine (VETOQUINOL). Each experiment wasperformed using 10 mice per group and was repeated three times.

5) Protection Experiments

The protective capacity of the antibodies was analysed using the murinemodel of pulmonary infection previously described (Voino et al., ActaMorpho., 1961, XI, 440-; Phalipon et al., J. exp. Med., 1995, 182,769-). Intranasal challenge was performed using either 10⁹ live virulentbacteria when protection was assessed by mortality assay or 10⁸ bacteriawhen protection was assessed by measurement of the lung-bacterial load.Naive mice were used as controls in each experiment. Mice immunized i.p.were challenged i.n. with virulent bacteria, 3 weeks after the lastimmunization. Mice passively transferred i.n. with polyclonal sera orwith purified mAbs were challenged 1 h after administration of the mAbs.Measurement of lung-bacterial load was performed at 24 h post infectionas follows. Mice were sacrificed by cervical dislocation and lungs wereremoved <<en bloc >> and ground in 10 ml sterile PBS (Ultra Turrax T25apparatus, Janke and Kunkel IKA Labortechnik GmbH). Dilutions were thenplated on Trypticase Soy Broth plates for c.f.u. enumeration. Eachexperiment was performed using 10 mice per group and was repeated threetimes.

5) Histopathological Studies

Mice were anesthesized, their trachea catheterized, and 4% formalininjected in order to fill the bronchoalveolar space. Lungs were thenremoved and fixed in 4% formalin before being processed forhistopathological studies. Ten-micrometer paraffin sections were stainedwith Hematoxiline and Eosin (HE), and observed with a BX50 Olympusmicroscope (Olympus Optical, Europa, GmbH).

6) Statistical Analysis

Significant differences were compared using the Student's test.Probability values <0.05 were considered significant.

B) Results

1) Protection conferred upon systemic immunization or intranasaladministration of specific immune serum.

In order to address the role of the systemic anti-LPS IgG antibodyresponse in protection against the mucosal infection, the protectionconferred against i.n. challenge with a lethal dose of S. flexneri 2abacteria in mice immunized i.p. with the homologous killed bacteria wasassessed. Antibodies induced upon such an immunization were mainlyanti-LPS IgG antibodies with all the IgG subclasses similarly elicited(FIG. 30A). No mucosal response was elicited, as reflected by theabsence of anti-LPS antibody response detectable in the bronchoalveolarlavage of immunized mice. Only 40% of the immunized mice survived thei.n. challenge, whereas 100% of naive mice succumbed. The low efficacyof systemic immunization in inducing protection could be due to eitherthe inability of anti-LPS IgG to be protective or the absence of theprotective antibodies (or their presence but in insufficient amount) inthe mucosal compartment at the time of i.n. challenge.

Therefore, it was tested whether the anti-LPS IgG antibodies may conferprotection if present locally prior to mucosal challenge. Polyclonalsera exhibiting different anti-LPS antibody titers were intranasallyadministered to naive mice 1 h prior to i.n. infection with a sublethaldose of S. flexneri 2a bacteria. Protection was assessed by thereduction of the lung-bacterial load in comparison to control mice andmice receiving preimmune serum. In contrast to control mice and micereceiving preimmune serum, naive mice receiving anti-LPS IgG serumshowed a significant decrease of the lung-bacterial load. The reductionwas dependent on the amount of anti-LPS IgG antibodies administered asreflected by the anti-LPS antibody titer of the immune serum used forpassive transfer. Thus, the highest reduction was obtained with serumhaving the highest anti-LPS antibody titer (1/64,000) (FIG. 30B, c);p=5×10⁻⁶ in comparison to mice receiving preimmune serum). However, inmice receiving immune serum with lower anti-LPS antibody titer (1/16,000and 1/4,000) (FIG. 30B, a and b), even if less efficient, the decreaseof the bacterial load was still significant in comparison to micereceiving preimmune serum (p=0, 027 and 0, 015, respectively).

These results demonstrated that, if present locally at the time ofmucosal challenge, the anti-LPS IgG antibodies were protective, thuslimiting bacterial invasion.

2) Protective capacity of different subclasses of mIgG specific for S.flexneri 2a LPS

Depending of the infecting strain, different subclasses of IgG specificfor LPS are induced following natural Shigella infection (Islam et al.,Infect. Immun., 1995, 63, 2045-2061). To test whether all subclassesexhibit similar protective capacity, murine mIgG specific for serotypedeterminants on the O—SP and, representative of each of the four murineIgG subclasses were obtained. Upon screening of hybridomas for theirreactivity with LPS from S. flexneri serotype X, Y, 5a, 5b, 2a, 2b, 1a,3a, respectively, five mIgG specific for S. flexneri 2a LPS wereselected: mIgG F22-4 (IgG1), mIgG D15-7 (IgG1), mIgG A2-1 (IgG2a), mIgGE4-1 (IgG2b) and mIgG C1-7 (IgG3). These hybridomas have been depositedon Apr. 20, 2004, at the “Collection National de Culture desMicroorganismes” from INSTITUT PASTEUR, 25 rue du Docteur Roux, 75724PARIS CEDEX 15, FRANCE, under the registration number I-3197, I-3198,I-3199, I-3200 and I-3201, for A2-1, C1-7, D15-7, E4-1 and F22-4,respectively.

The avidity of each mIgG for LPS, defined by IC₅₀, ranged from 2 to 20ng/ml. To analyse the protective capacity of the selected mAbs, naivemice were administered i.n. with each of the purified mIgG prior to i.n.challenge with a S. flexneri sublethal dose. Upon challenge,lung-bacterial load in mice passively administered with 20 μg of each ofthe mIgG specific for S. flexneri 2a LPS was significantly reduced incomparison to mice receiving PBS (FIG. 31A). Upon passive transfer using2 μg of mIgG, only mIgG D15-7, A2-1 and E4-1 were shown to significantlyreduce the lung-bacterial load in comparison to control mice, but withmuch less efficiency than that observed using 20 μg (FIG. 31A). As shownin FIG. 31B, reduction of lung-bacterial load in mice receiving 20 μg ofmIgG was accompanied by a reduction of inflammation and therefore ofsubsequent tissue destruction. In comparison to control mice showing anacute broncho-alveolitis with diffuse and intense polymorphonuclear cellinfiltration (FIG. 31B, a, b) associated with tissular dissemination ofbacteria (FIG. 2B, c), only restricted areas of inflammation wereobserved in antibody-treated mice, essentially at the intra- andperibronchial level (FIG. 31B, d, e), where bacteria localized (FIG.31B, f). Following passive administration with 2 μg of mIgG,inflammation resembled that of the control mice with a similar patternof Polymorphonuclear (PMN) infiltration and tissue destruction, inaccordance with the very low, if any, reduction in lung-bacterial load.

These results with murine monoclonal antibodies (mAbs) of the G isotype(mIgG) representative of the different IgG subclasses and specific forserotype-specific determinants on the O—SP, demonstrated that each IgGsubclass exhibited a similar serotype-specific protective capacity, withsignificant reduction of the lung-bacterial load and of subsequentinflammation and tissue destruction. These antibodies may conferprotection by different pathways involving or not the complementcascade. In the present study, all the different murine IgG subclasseswere shown to be protective, suggesting that depending on the subclass,different mechanisms may be involved in IgG-mediated protection. Whereasantibody-dependant cellular cytotoxicity (ADCC) has been reported forShigella-specific secretory IgA and lymphocytes from the gut-associatedlymphoid tissues (Tagliabue et al., Nature, 1983, 306, 184-186),Shigella IgG-mediated ADCC occurs in vitro with splenic T cells but notwith T lymphocytes from the GALT (Tagliabue et al., J. Immunol., Nature,1984, 133, 988-992). Further studies using mice deficient for T cells orfor proteins of the complement cascade will be required to analyze theIgG-mediated protective mechanisms in vivo.

3) Serotype-Specific Protection Induced by the Anti-LPS mIgG

Antibodies specific for epitopes common to several serotypes of a givenspecies as well as serotype-specific antibodies are elicited uponnatural or experimental infection (Rasolofo-Razanamparany, Infect.Immun., 2001, 69, 5230-5234, Van de Verg et al., Vaccine, 1996, 14,1062-1068). However, the serotype-specific protection observed followingnatural or experimental infection suggests that the antibodies directedagainst serotype determinants play a major protective role (Du Pont etal., J. Infect. Dis., 1972, 125, 12-; MeI et al., Bull. W.H.O., 1968,39, 375-380). For instance, mIgA specific for S. flexneri serotype 5ahas been shown to protect only against homologous challenge (Phalipon etal., J. Exp. Med., 1995, 182, 769-). Therefore, it was tested whetherthe protection observed with the anti-LPS mIgG obtained in this studywas also serotype-specific. Mice passively administered with 20 μg ofmIgG C1 specific for S. flexneri 2a were protected against homologouschallenge, but not upon heterologous challenge with S. flexneri 5abacteria (FIG. 32A). Similarly, mice receiving 20 μg of mIgG C20, a mAbspecific for S. flexneri serotype 5a and, of the same isotype than mIgGC1, i.e. IgG3, showed a significant reduction of lung-bacterial loadupon i.n. challenge with S. flexneri 5a, but not with S. flexneri 2a(FIG. 32A). In mice protected against homologous challenge, inflammationwas dramatically reduced with a slight intra- and peribronchial PMNinfiltrate remaining present (FIG. 32B, b and c). In contrast, in micenot protected upon heterologous challenge (FIG. 32B, a and d),inflammation and tissue destruction were similar to those observed incontrol mice (FIG. 32B, and b).

The protective role of the serotype-specific antibody response has beenfirstly emphasized in a study using a monoclonal dimeric IgA (mIgA)specific for a S. flexneri serotype 5a determinant (Phalipon et al., J.Exp. Med., 1995, 182, 769-51). The results presented here demonstratethat mIgGs specific for S. flexneri serotype 2a or serotype 5a alsoconfer serotype-specific protection. It seems that whatever the antibodyisotype and the bacterial strain, the serotype-specific antibodyresponse is protective against homologous bacterial challenge. It shouldbe noted that using the same amount of mIgA and mIgG specific for S.flexneri 5a, both exhibiting a similar IC₅₀ for LPS, reduction inlung-bacterial load was much more efficient with mIgA. Actually, incontrast to mIgG, protection was observed in the presence of 2 μg ofmIgA. The discrepancy between the two isotypes may be due to thedimeric/polymeric (d/p) form of mIgA, which mimics the IgA response atthe mucosal surface. In contrast to monomeric IgG, interaction of d/pIgA exhibiting at least four antigen-binding sites with a specificdeterminant highly repeated on the bacterial O—SP surface may lead tothe formation of aggregates that are efficiently removed by localphysical mechanisms (Corthésy et al., Curr. Top. Microbiol. Immunol.,1999, 236, 93-111). Also, quantitative assessment of IgG and IgAsubclass producing cells in the rectal mucosa during shigellosis inhumans has revealed the predominance of the IgA response. The IgGresponse which is about 50 times lower than the IgA response is mainlyIgG2 and correlates with the presence of specific IgG2 in serum. Thiscorrelation suggests that the majority of the Shigella specific serumantibodies are derived from the rectal mucosa (Islam et al., J. Clin.Pathol., 1997, 50, 513-520). Together, these results suggest that in thesituation where both local and systemic anti-LPS antibody responses areinduced, as for example upon natural infection, the local SIgA-mediatedresponse will be the major protective response, with the IgG-mediatedresponse possibly contributing to a lesser extent to local protection.

On the other hand, the data presented here suggest that in the absenceof local SIgA-mediated response, as for example upon vaccination via thesystemic route using glycoconjugate vaccines, the systemic anti-O—SPresponse induced is effective in protecting against homologous Shigellainfection, if the effectors are present locally. Previous reports haveshown that serum IgGs may protect from gastrointestinal infections(Bougoudogo et al., Bull. Inst. Pasteur, 1995, 93, 273-283; Pier et al.,Infect. Immun., 1995, 63, 2818-2825). Therefore, it should be admittedthat serum IgG efficiently gain access to the intestinal barrier inorder to prevent bacterial invasion and dissemination. How IgG crossesthe epithelial barrier to function in mucosal immunity remains unclear.One possible pathway is passive transudation from serum to intestinalsecretions (Batty et al., J. Pathol., Bacteriol., 1961, 81, 447-458;McCleery et al., Digestion, 1970, 3, 213-221; Wernet et al., J. Infect.Dis., 1971, 124, 223-226). After its passage of the intestinal barrierthrough M cells and its interaction with resident macrophages andepithelial cells, Shigella initiates an inflammatory response leading toinfiltration of the infected tissues with polymorphonuclear cells(Philpott et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 2000, 29,575-586). It may therefore be reasonably envisioned that specific serumIgGs transudate to the intestinal tissue during this inflammatoryprocess that occurs very soon after bacterial translocation. Anotherexplanation could be the involvement of the FcRn receptor in IgGtransport. FcRn was firstly identified as the Fc receptor responsiblefor transferring maternal IgGs from mother's milk across the intestinalEC of the neonatal gut of rodents. Much evidence supports the conceptthat FcRn is ubiquitously expressed in adult tissues and plays a role inIgG homeostasis, dealing with IgG half-life (Ghetie et al., Ann. Rev.Immunol., 2000, 18, 739-766). It has been recently reported that thisreceptor is expressed by enterocytes in human adults and mediatestranscytosis of IgG in both direction across the intestinal epithelialmonolayer (Ramaligan et al., EMBO J., 1997, 21, 590-601). Furtherinvestigation is required to improve the knowledge on the role played byFcRn in IgG-mediated protection of the intestinal barrier againstenteropathogens. Nevertheless, the existence of such a pathway alreadyenlarges the current view of the humoral response at mucosal surfaces.

4) Absence of Protection Induced by the mIgG Specific for S. flexneriInvasins

The invasins IpaB and IpaC are essential to the expression of theShigella invasive phenotype (Ménard et al., J. Bacteriol., 1993, 175,5899-5906). Moreover, they are targets for the humoral response sinceantibodies specific for both proteins are detected in sera of patientsconvalescent from shigellosis (dam et al., J. Clin. Microbiol., 1993,31, 454-457; Oaks et al., Infect. Immun., 1986, 53, 57-63; Oberhelman etal., Infect. Immun., 1991, 59, 2341-2350; Van de Verg et al., J. Infect.Dis., 1992, 166, 158-161). To assess whether the anti-invasin antibodyresponse may contribute to protection, in addition to the anti-LPSantibody response, mIgG recognizing different epitopes on IpaB or IpaC,were used (Barzu et al., Infect. Immun., 1993, 61, 3825-3831; Phaliponet al., Infect. Immun., 1992, 60, 1919-1926). Whatever the dose used, incontrast to mIgG C20, no reduction in lung-bacterial load was measuredupon challenge in mice treated with mIgG H16 and mIgG H4 recognizingdistinct epitopes in the central region of IpaB or with mIgG J22 andmIgG K24 recognizing the N- and the C-termini domain of IpaC,respectively (FIG. 33). Protection was also not observed upon combininganti-IpaB and anti-IpaC mIgG.

The results presented here demonstrated that mIgG specific for IpaB orIpaC are not protective despite the fact that they are directed againstepitopes located in different regions of these proteins (Barzu et al.,Infect. Immun., 1993, 61, 3825-3831; Phalipon et al., Infect. Immun.,1992, 60, 1919-1926) and that they have been shown to interfere withtheir functional properties in in vitro studies (Barzu et al., Infect.Immun., 1998, 65, 1599-1605; Ménard et al., Cell, 1994, 79, 515-525).The most likely explanation is that these invasins, that are secretedthrough the type III secretion apparatus, are injected straight into thehost cell, upon contact of the bacterium with the cell membrane (Ménardet al., EMBO J., 1994, 13, 5293-5302; Blocker et al., Mol. Microbiol.,2001, 39, 652-663). Therefore, there is probably very limited access, ifany, for specific antibodies to interact with their targets. Althoughnot tested, it is unlikely that the local SIgA-mediated response tothese proteins will be protective.

III Characterization of the Serotype-Specific Antigenic Determinants ofS. Flexneri Serotype 2a Lipopolysaccharide

Antigenic determinants recognized by protective monoclonal antibodieswere characterized in a competition ELISA using synthetic di-, tri-,tetra- and pentasaccharides obtained by circular permutation of theresidues from the repetitive units of the O—SP from S. flexneri serotype2a (FIG. 29), as well as longer fragments (octa- and deca-saccharides),as competitors for binding of the antibodies to the homologous LPS.

A) Material and Methods

1) Synthetic Oligosaccharides Representative of S. flexneri Serotype 2aO—SP

Oligosaccharides representative of fragments of the O—SP of S. flexneri2a were synthesized by multistep chemical synthesis, as described in thepreceding examples.

TABLE A: Oligosaccharides* representative of fragments of the O-SP of S.flexneri 2a Disaccharide Trisaccharide Tetrasaccharide PentasaccharideOctasaccharide Decasaccharide AB ABC BC BCD DA CDA (E)C DAB (βE)C B(E)CAB(E)C DAB(E)C {DAB(E)C}₂ (E)CD B(E)CD B(E)CDA B(E)CDAB(E)C A(βE)C(E)CDA (E)CDAB AB(E)CD

The oligosaccharides were synthesized as methyl glycoside in order tomimic the glycosidic linkages present in the natural polysaccharide andprevent any ambiguity which may otherwise arise due to equilibriummixtures of the α- and β-anomers corresponding to the furanose andpyranose forms of the reducing residue.

The βEC and A(βE)C compounds, which have a non natural EC glycosidiclinkage, were synthesized in order to probe the influence of suchlinkage on Ab recognition. Since they were estimated to be the easiestchemically accessible targets, the octa-B(E)CDAB(E)C and decasaccharideDAB(E)CDAB(E)C were chosen as the longer fragments in order to gain someknowledge on the length-dependent oligosaccharide-antibody recognition.

2) Monoclonal Antibodies

The monoclonal antibodies specific for serotype 2a used in this studyare the five IgG antibodies described in example X+1: F22-4, D15-1,E4-1, A-2, and C1-7. In addition, an IgG monoclonal antibody specificfor serotype 5a (C20) was used as control.

3) Inhibition ELISA.

First of all, a standard curve was established for each antibody tested.Different concentrations of the antibody was incubated at 4° C.overnight and then incubated on microtiter plates coated with purifiedShigella flexneri LPS homologous to the strain used for the obtention ofthe antibody, at a concentration of 5 μg/ml in carbonate buffer at pH9.6, and previously incubated with PBS/BSA 1% for 30 min at 4° C. Afterwashing with PBS-Tween 20 (0.05%), alkaline phosphatase-conjugatedanti-mouse IgG was added at a dilution of 1:5000 (Sigma Chemical CO.)for 1 h at 37° C. After washing with PBS-Tween 20 (0.05%), the substratewas added (12 mg of p-nitrophenylphosphate in 1.2 ml of Tris, HCl bufferph 8.8 and 10.8 ml of NaCl 5M). Once the color developed, the plate wasread at 405 nm (Dinatech MR 4000 microplate reader). A standard curveOD=f(antibody concentration) was fitted to the quadratic equationY=aX²+bX+c where Y is the OD and X is the antibody concentration.Correlation factor (r²) of 0.99 were routinely obtained.

Then, the amount of oligosaccharides giving 50% inhibition of IgGbinding to LPS (IC₅₀) was then determined as follows. IgG at a givenconcentration (chosen as the minimal concentration of antibody whichgives the maximal OD on the standard curve) was incubated overnight at4° C. with various concentrations of each of the oligosaccharides to betested, in PBS/BSA 1%. Measurement of unbound IgG was performed asdescribed in the preceding example, using microtiter plates coated withpurified LPS from S. flexneri 2a and the antibody concentration wasdeduced from the standard curve. Then, IC₅₀ was determined.

4) mIgG Sequence Analysis

Total RNA was extracted from hybridoma cells by RNAxeI kit (EUROBIO).mRNA was converted into cDNA with a reverse transcriptase kit(INVITROGEN) and used as template for PCR amplification using Taq DNApolymerase (GIBCO, BRL) according the manufacturer's protocol. Theamplification was performed with the primer of corresponding isotype(SEQ ID NO: 1 to 3; IgG1: 5′ GCA AGG CTT ACT AGT TGA AGA TTT GGG CTC AACTTT CTT GTC GAC 3′; IgG2a: 5′ GTT CTG ACT AGT GGG CAC TCT GGG CTC 3′;IgG3: 5′GGG GGT ACT AGT CTT GGG TAT TCT AGG CTC 3′. The following eightheavy chain variable region (VH) primers were also used (SEQ ID NO: 4 to11:5′ GAG GTG CAG CTC GAG GAG TCA GGA CC3′; 5′ GAG GTC CAG CTC GAG CAGTCT GGA CC 3′; 5′ CAG GTC CAA CTC GAG CAG CCT GGG GC 3′; 5′ GAG GTT CAGCTC GAG CAG TCT GGG GC 3′; 5′ GAG GTG AAG CTC GAG GAA TCT GGA GG 3′; 5′GAG GTA AAG CTC GAG GAG TCT GGA GG 3′; 5′ GAA GTG CAG CTC GAG GAG TCTGGG GG 3′; 5′ GAG GTT CAG CTC GAG CAG TCT GGA GC 3′). Nucleic acidsequences were carried out by GENOME EXPRESS S.A. using PCR products.Sequence analysis was performed with software package from the GeneticsComputer Group, Inc (Madison, Wis.), the Genebank (Los Alamos, N. Mex.)and EMBL (Heidelberg, Germany) databases. For the determination of thegenes families, analysis of the nucleotide sequences was performed withthe international ImMunoGeneTics database (Lefranc, M.-P., 2003 NucleicAcids Res., 31,307-310).

TABLE B: Minimal sequence recognized by the mIgG F22-4 D15-7 A2-1 E4-1C1-7 IgG1 IgG1 IgG2a IgG2b IgG3 Motif IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ (*)(μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L)CD >1000 >1000 >1000 >1000 >1000 EC >1000 >1000 >1000 >1000 >1000B(E)C >1000 >1000 >1000 >1000 >1000 (E)CD 179 >1000 >1000 >1000 >1000(E)CDA 181 >1000 >1000 >1000 >1000 (E)CDAB 354 >1000 >1000 >1000 >1000B(E)CD 5 198 >1000 87 >1000 B(E)CDA 2.5 240 350 75 400AB(E)C >1000 >1000 >1000 >1000 >1000DAB(E)C >1000 >1000 >1000 >1000 >1000 AB(E)CD 21 490 378 287 734 (*)Oligosaccharides are methyl glycosides derivatives

None of the mono- or disaccharides showed any binding when used at aconcentration of 1 mM. Evaluation of trisaccharide recognition outlinedthe unique behaviour of mIgG F22-4, which was the only Ab showingmeasurable affinity for such short oligosaccharides. ECD was the onlytrisaccharide recognized by F22-4, pointing out the crucial contributionof both the branched glucosyl residue (E) and the N-acetyl-glucosaminylresidue (D) to Ab recognition. This was supported by the absence ofrecognition of AB(E)C or DAB(E)C by none of mIgG. Comparison of therecognition of the branched tetrasaccharide B(E)CD to that of the linearECD indicated that rhamnose B, accounting for an improvement of the IC₅₀by a factor of ˜50, was also a key element in the Ab recognition.Indeed, B(E)CD was recognized by all the protective mIgG, except A2-1and C1-7 for which the minimal sequences necessary for recognition werepentasaccharides AB(E)CD or B(E)CDA. Extension of B(E)CD at the reducingend, yielding the branched pentasaccharide B(E)CDA, did not result inany major improvement of Ab binding for the other mIgGs. The minor, ifnot absent, contribution of reducing A to binding was also apparent whencomparing recognition of ECD and ECDA by F22-4. Further elongation atthe reducing end, yielding ECDAB did not improve binding to F22-4.Introduction of residue A at the non reducing end of B(E)CD, leading toAB(E)CD, had a somewhat controversial impact on Ab recognition with apositive effect in the case of A2-1, and only a slight effect in thecase of C1-7, and even negative by a factor ˜2 to ˜5 when consideringthe other antibodies. Therefore, for the recognition of shortoligosaccharides, two families of mIgGs were identified. The first onerepresented by F22-4 recognizing the ECD trisaccharide, and the secondone, comprising the remaining four mIgGs, that recognized the samecommon ECD sequence flanked by the B residue at the non reducing end,added or not with A residue at the non reducing or reducing end.

This observation was confirmed when measuring the recognition of longeroligosaccharides (Table C).

TABLE C Antibody recognition is improved with longer oligosaccharides(*) B(E)CDA AB(E)CD B(E)CDA B(E)C DA B(E)CDA B(E)C AnticorpsIC₅₀(μmol/L) IC₅₀(μmol/L) IC₅₀(μmol/L) IC₅₀(μmol/L) F22-4 (IgG1) 2.521.6 0.22 5 D15-7 (IgG1) 240 490 60.8 11.9 A2-1 (IgG2a) 350 378 12.9 3E4-1 (IgG2b) 75 287.7 12 4.4 C1-7 (IgG3) 400 734 242 19 (*) Alloligosaccharides are methylglycosides derivatives

Indeed, the decasaccharide was the highest affinity ligand for allantibodies except F 22-4. In the latter case, the octasaccharide was thebest recognized sequence with an IC₅₀ of 0.22 μM, corresponding to animprovement by a factor ˜10, when compared to pentasaccharide B(E)CDA.Further extension of the octasaccharide by addition of DA at the nonreducing end resulted in a loss of recognition by a factor of ˜20.Interestingly, the recognition of these two longer oligosaccharides bythe other mIgGs differed from that of F22-4. D15-7 and E4-1 behavedsimilarly, with extension by B(E)C at the reducing end leading to theoctasaccharide, and then by DA at the non reducing end, leading to thedecasaccharide, both resulting in improving Ab binding by a factor of˜4. C1-7 behaved somewhat differently since contribution of B(E)C tobinding appeared to be minor, whereas introduction of DA, resulted, asfor the above cited mIgG, in an overall gain in binding of ˜20. Finally,in the case of A2-1, addition of B(E)C to the reducing end ofpentasaccharide B(E)CDA resulted in a gain in recognition by a factor˜25, and subsequent addition of DA at the non reducing end furthercontributed to binding improvement by a factor of ˜4. To summarize,lengthening the oligosaccharide sequence improved the Ab recognition.

Thus, the data presented indicate the presence of an immunodominantepitope (E)CD of S. flexneri serotype 2a lipopolysaccharide, withflanking residues contributing to the recognition depending on themonoclonal antibody. The sequences B(E)CDA and AB(E)CD are almostsimilarly recognized by all the monoclonal IgG antibodies. In addition,the recognition improvement observed with longer oligosaccharidesindicate that multiple epitopes along the polysaccharide chain (17repetitive units in average) but not one unique epitope at theextremity, are presented on the LPS.

2) Molecular Characterization of the Protective S. flexneri Serotype2a-Specific mIgG

To analyse whether the differences observed in the recognition ofoligosaccharides by the mIgGs reflect differences in the structure ofthese mAbs, their complementary-determining regions (CDRs) weresequenced (Table D and E).

TABLE D VH domain CDR sequences CDR1 (SEQ CDR3 OLIGO- ID NO: CDR2(SEQ ID SAC- 12 to (SEQ ID NO: NO: CHARIDE VH 15 16 to 19) 20 to 23)MOTIF F22-4 NYWMS EIRLKSDNYATYYAESVKG PMDY ECD D15-7 YSSIHWINTATGEPTYPDDFKG YDYAGFYW B(E)CD A2-1 DYSLH WINTETGEPAYADDFKG YRYDGAYB(E)CDA E4-1 DYSMH WVNTQTGEPSYADDFKG YRYDGAH B(E)CD C1-7 B(E)CDA

TABLE E VL domain CDR sequences CDR2 CDR3 OLIGO- CDR1 (SEQ ID (SEQ IDSAC- (SEQ ID NO: NO: 28 NO: CHARIDE VL 24 to 27) to 31) 32 to 34) MOTIFF22-4 RSSKSLLHSDGITYLY HLSNLAS AHNVELPRT ECD D15-7 SASSSVGYIH DTSKLASQQWSRNPLT B(E)CD A2-1 RATSSVGYIN ATSNLAA QQWSSDPFT B(E)CDA E4-1RARSSVGYM ATSNQAS QQWSSDPFT B(E)CD C1-7 B(E)CDA

Only two VH and Vκ gene families were expressed among the five studiedmIgG (Table F).

TABLE F: V gene usage mAb isotype VH D JH VK JK  A2-1 IgG2a VGAM3-8 SP2JH3 VK4/5 JK4  C1-7 IgG3 D15-7 IgG1 VGAM3-8 SP2 JH3 VK4/5 JK5  E4-1IgG2b VGAM3-8 SP2 JH3 VK4/5 JK4 F22-4 IgG1 J606 not known JH4 VK24/25JK1

VH J606 (Brodeur et al., Eur. J. Immunol., 1984, 14, 922-930) andVK24/25 (Almagro et al., Immunogenetics, 1998, 47, 355-363) encodedF22-4 VH and Vκ, respectively. A2-1, D15-7 and E4-1 VH genes weremembers of the VGAM3-8 family (Winter et al., Embo J., 1985, 4,2861-2867) and their Vκ genes belonged to the VK4/5 family (Almagro etal., precited). The joining segment of F22-4 heavy chain was encoded byJH4 (Sakano et al., Nature, 1980, 86, 676-683), while A2, D15-7 and E4-1heavy chains shared the same diversity and joining segments, DSP2 (Gu etal., Cell, 1991, 65, 47-54) and JH3 (Sakano et al., precited),respectively. The joining segment for the light chain is encoded by JK1(Max et al., J. Biol. Chem., 1981, 256, 5116-5120) for F22-4, JK4 for A2and E4-1, and JK5 for D15-7. The four antibody CDRs except for CDRH3,fall into the canonical structure classes (Al-Lazikani et al., J. MolBiol, 1997, 273, 927-948). For all mIgG, the CDRs L2, L3 and H1 were ofthe same classes, 1/7A, 1/9A and 1/10A, respectively (Martin et al., J.Mol. Biol., 1996, 263, 800-815). For F22-4, the canonical form of theloops L1 and H2 were of the classes 4/16A and 4/12A, while those of thethree other antibodies fall into classes 1/10A for L1 and 2/10A for H2.The CDR-H3 of A2, D15-7 and E4-1 contained seven residues along withseveral aromatic ones, while the CDR-H3 of F22-4 was very short, onlyfour amino-acids with a proline residue in the first position.

mIgG F22-4 binds to the O—SP in an unique mode, selecting the lineartrisaccharide ECD as the minimal sequence necessary for recognition at aconcentration below 1 mM. The specificity of F22-4 suggests that theglucose residue (E) is probably involved in direct interactions with theAb, while for the other mAbs, E may also constrain the conformation ofanother part of the oligosaccharide that interacts with the Ab. F22-4uses a VHJ606/VK24/25 pair. The J606 family comprises VH genes encodingthe immune response to β-(1,6)-galactan (Hartman et al., 1984, 3,2023-2030). The CDRs H1, H2, L1 and L2 are quite similar in sequenceand/or length to those of SYA/J6 (Table G), a mAb generated in responseto immunization with S. flexneri Y.

TABLE G Comparison of the sequenccs of SYA/J6 (SEQ IDNO: 12, 35 to 39) and F22-4 (SEQ ID NO: 12, 16, 20, 24, 28 and 32) CDRs*VH H1 H2 H3 31 35 52abc 100a SYA/J6 NYWMS EIRLKSNNYATHYAESVKG GGAVGAMDYF22-4 NYWMS EIRLKSDNYATYYAESVKG PMDY VH L1 L2 L3 27abcde 30 50   5689    97 SYA/J6 RSSQSLLHSDGNTYLH KVSNRFS SQTTHVPT F22-4 RSSKSLLHSDGITYLYHLSNLAS AHNVELPRT *Kabat numbering

In contrast, the H3 loops, which are the major key of Ab diversity, arevery different. In mAb SYA/J6, the CDR-H3 comprises nine amino-acids;its base which possesses three Gly residues, shows the torso-bulgedstructure (Morea et al., J. Biol. Chem., 1998, 263, 269-294) and thismAb is an example of a groove like site for binding an internaloligosaccharide epitope (Vyas et al., Biochemistry, 2002, 41,13575-13586). In the case of F22-4, the H3 loop-four residues, which canonly form a short hairpin, would allow a more open binding site, thancan accommodate the linked glucose.

The improved F22-4 recognition of the tetrasaccharide B(E)CD outlinesthe key input on the branching site. However, as found in the case ofpentasaccharide AB(E)CD and decasaccharide DAB(E)CDAB(E)C, furtherextension at the non reducing end of this key fragment had a negativeimpact on binding. These findings suggest that although the Ab combiningsite is most probably of the groove type, it is somewhat restricted onone side and unable to accommodate inappropriate extension.

The other mIgGs require B(E)CD as the minimal sequence recognized at aconcentration below or close to 1 mM (A2-1). These mAbs probably bindintrachain epitopes, as it is supported by the fact that the longer theoligosaccharide, the better the recognition. It is somewhat puzzling tonote that although binding to the shorter oligosaccharides is slightlydifferent, all the mIgGs fall into the same pattern of affinity whenconsidering the decasaccharide. The most striking observation concernsA2-1, for which a 100 fold increase in binding was noted when comparingDAB(E)CDAB(E)C to B(E)CDA. It is noteworthy that these mIgGs use aVGAM3-8/VK24/25 pair, thus differing from F22-4. The VGAM3.8 multigenefamily was isolated from the DNA of mouse B-lymphocytes stimulated byLPS (Winter et al., Embo J., 1985, 4, 2861-22867).

Taken together, these results suggest that the particular behaviour ofF22-4 in recognizing of the trisaccharide ECD, in comparison to theother mIgGs, could be related to particular molecular structure.

IV. PREPARATION OF TT CONJUGATES

a) Material and Methods

N-(γ-maleimidobutiryloxy) sulfosuccinimide ester (sulfo-GMBS) waspurchased from Pierce. Tetanus toxoid (TT) (MW 150 kDa) (batch n^(o)FA045644), was purchased from Aventis Pasteur (Marcy l'Etoile, France),and stored at 4° C. in a 39.4 mg·mL⁻¹ solution.

Dialyses were performed with Slide-A-Lyzer® Dialysis Cassettes (Pierce)and concentration by centrifugation using Vivaspin 15R centrifugalconcentrators (Vivascience, Palaiseau, France), displaying a membranecut-off of 10000 Da, at a centrifugal force of 4500×g.

i) pmLPS-TT Conjugates

Preparation and Derivatization of S. flexneri 2a pmLPS

S. flexneri 2a LPS was treated with acetic acid to hydrolyse the lipidA-core linkage: LPS [10 mg in 1% (v/v) aqueous acetic acid (1 mL)], washeated at 100° C. for 60 min. Precipitated lipid A was removed bylow-speed centrifugation (350×g for 15 min) at 4° C. The supernatant wasextracted with equal volume of chloroform-ethanol (2:1). The reactionmixture was shaken vigorously and centrifuged at 10,000×g for 60 min at4° C. The aqueous phase was dialyzed against distilled water to removeethanol and then freeze-dried to give S. flexneri 2a pmLPS (5.3 mg,53%).

S. flexneri 2a pmLPS (2.2 mg, 0.13 μmol) was dissolved in water (430 μL)at an actual concentration of 5 mg·mL⁻¹. The solution was brought to pH11 with 2 N NaOH, and an equal weight of CNBr (4.0 μL of a 5 M solutionin CH₃CN) was added. The pH was maintained at 11 with 2 N NaOH for 6 minat rt. An equal volume of adipic acid di-hydrazide 430 μL of a 0.5 Msolution in 0.5 M NaHCO₃) was added, and the pH was adjusted to 8.5 with0.5 M HCl. The reaction mixture was kept overnight at 6° C. and dialyzedagainst 0.1 M potassium phosphate buffer at 4-6° C.

The extent of derivatization of the activated pmLPS was calculated asthe ratio of adipic acid dihydrazide/polysaccharide (w/w) and foundequal to 3.7% using trinitrobenzenesulfonic acid (TNBS), as titrationreagent (Habeeb, A. F., Anal. Biochem., 1966, 14, 328-336).

Preparation and Characterization of the Conjugate

The activated S. flexneri 2a pmLPS (1.8 mg), and the succinic anhydridetreated TT (1.8 mg) were mixed. Solid1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (5.3 mg), was thenadded to a final concentration of 0.1 M and the pH of the reactionmixture was maintained at 6 for 4 h at rt. The crude mixture wasdialyzed against PBS 1×(3×2 L) at 4-6° C. and passed trough a CL-6BSepharose column (1 m×160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH7.4 as eluent at a flow rate of 0.2 mL·min⁻¹, with detection bymeasuring the optical density at 280 nm and the refractive index. Thefractions containing the conjugates were pooled and concentrated. Theconjugate was stored at 4° C. in the presence of thimerosal (0.1mg·mL⁻¹) and assessed for its total carbohydrate and protein content.

ii) Oligosaccharide-TT Conjugates

Derivatization of TT

In a representative example, to a solution of TT (12 mg, 304 μL, 0.08μmole) diluted in 0.1 M PBS, pH 7.3 (296 μL), was addedN-(γ-maleimidobutiryloxy) sulfosuccinimide ester (GMBS) (3×1.53 mg, 3×58μL of an 30 mg·mL⁻¹ solution in CH₃CN, 3×50 equiv), in three portionsevery 40 minutes. The pH of the reaction mixture was controlled(indicator paper) and maintained at 7-7.5 by addition of 0.5 M aq NaOH.Following an additional reaction period of 40 minutes, the crudereaction mixture was dialyzed against 3×2 L of 0.1 M potassium phosphatebuffer, pH 6.0 at 4° C. to eliminate excess reagent. About 45 maleimidegroups were introduced on TT as indicated by SELDI-TOF MS analysis.

Conjugation

Following dialysis, maleimide activated-TT in 0.1 M potassium phosphatebuffer solution was divided into several portions which were furtherreacted with synthetic S-acetylthioacetylated-tri-, tetra- penta-,hexa-; deca- and pentadecasaccharides related to S. flexneri 2a O—SP ina 1:12 molar ratio, respectively. Reaction mixtures were buffered at a0.5 M concentration by addition of 1 M potassium phosphate buffer, pH6.0. Then, NH₂OH, HCl (7.5 μL of a 2 M solution in 1 M potassiumphosphate buffer, pH 6), was added to the different mixtures and thecouplings were carried out for 2 h at rt. The conjugated products weredialyzed against 3×2 L of 0.05 M PBS, pH 7.4 at 4° C., and furtherpurified by gel permeation chromatography on a sepharose CL-6B column (1m×160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at aflow rate of 0.2 mL·min⁻¹, with detection by measuring the opticaldensity at 280 nm and the refractive index. The fractions containing theconjugates were pooled and concentrated. The conjugates were stored at4° C. in the presence of thimerosal (0.1 mg·mL⁻¹) and assessed for theirtotal carbohydrate and protein content.

In an attempt to maximize the loading of the protein, the derivatized-TTwas reacted as described above but in a 1:56 molar ratio using thepentadecasaccharide related to S. flexneri 2a O—SP.

Hexose concentrations were measured by a colorimetric method based onthe anthrone reaction, using pmLPS as a standard.

Protein concentrations were measured by the Lowry's spectrophotometricmethod, using BSA as a standard and/or total acidic hydrolysis (6 N HClat 110° C. for 20 h), using norleucine as an internal standard.

Determination of Hexoses with Anthrone

Reagents: The Reagents are as Follows

Stock sulfuric acid. Add 750 mL of concentrated sulfuric acid to 250 mLof distilled water and cool the solution to 4° C.

Anthrone reagent. Dissolve 1.5 g of anthrone in 100 mL of ethyl acetateand cool the solution to 4° C.

Standard oligosaccharide solution: Prepare a solution at a concentrationof 4 mg·mL⁻¹ in water. Prepare serial dilutions of 400 to 25 μMol of atetra- or pentasaccharide [B(E)CD and AB(E)CD, respectively] standardsolution in water. The tetra- and pentasaccharide standard solutionswere used to dose the conjugates obtained using tri-, tetra-, penta- orhexa-, deca- and pentadecasaccharide, respectively.

Procedure:

Prepare serial dilutions of 400 to 25 μMol of the appropriateoligosaccharide standard solution in water (1 mL) in screw-threadedtubes. Prepare similarly a reagent blank containing 1 mL water andcontrol reagents containing a known amount of pmLPS of S. flexneri 2aO—SP or glucose in 1 mL water. Prepare samples and make up to 1 mL ifnecessary by adding water. Cool all tubes in ice-water.

To each tube, add 5 mL of the concentrated H₂SO₄ and 0.5 mL of theanthrone solutions. Heat the tubes at 100° C., caps unscrewed for 3minutes and then caps screwed for 7 minutes. After exactly 10 minutes,return the tubes to an ice-bath and when cool measure the absorbance ina spectrophotometer (Seconam S.750I), at a wavelength of 625 nm. Thequantity of carbohydrate in the unknown samples can be read off from thestandard curve prepared with the standard solution samples and theblank.

b) Results

Characteristics of representative conjugates are listed in Table L.

TABLE L Iso- carbohydrate/ Conjugate lated protein Hapten/proteinreference Hapten yield wt/wt (%) (mmol/mmol) CGS0303-8-3 (E)CD 70% 5.412 CGS0303-8-4 B(E)CD 64% 7.4 13.3 CGS0303-8-5 AB(E)CD 80% 9.6 14.7CIMG745 B(E)CD 66% 6.5 10.8 CIMG746 AB(E)CD 85% 6.5 10.9 CGS0703-56-10[AB(E)CD]₂ 71% 16 13.5 CGS0703-56-15 [AB(E)CD]₃ 67% 43 24 CGS0104-113-4B(E)CD 52% 12 15.8 CGS0104-113-5 AB(E)CD 51% 10 13 CGS0104-113-6DAB(E)CD 72% 13 17 CGS0104-113-10 [AB(E)CD]₂ 62% 22 14 CGS0104-113-15[AB(E)CD]₃ 68% 4 26 CGS0204-121 pmLPS 88% 41 3.6^(a) CGS0703-51 pmLPS74% 25 2.2^(a) ^(a)Based on an estimated Mr of 17,000 kD for pmLPS(pmLPS stands for LPS detoxified by acid hydrolysis)

V Immunogenicity of the Oligosaccharides-Tetanus Toxoid Conjugates

A) Material and Methods

1) Immunization Protocol

Two immunization assays in the absence of adjuvant were performed witholigosaccharides conjugated to tetanus toxoid, prepared as described inpreceding example.

In a first assay, groups of eight mice received four intramuscularinjections at three weeks interval of B(E)CD, AB(E)CD, DAB(E)CD,[AB(E)CD]₂ or [AB(E)CD]₃ oligosaccharides conjugated to tetanus toxoid(10 μg oligosaccharide/mice/injection). Control mice received detoxifiedLPS from S. flexneri 2a conjugated to tetanus toxoid (10 μgpolysaccharide/mice/injection) by multipoint attachment, as described byTaylor et al., Infect. Immun., 1993, 61, 3678-3687, or tetanus toxoidalone (140 μg/mice/injection), following the same immunization schedule.One month after the last injection, the mice received a last boost ofconjugates, in the same conditions.

In a second assay, groups of seven mice received three intramuscularinjections at three weeks interval of B(E)CD, DAB(E)CD, and groups offourteen mice received three intramuscular injections at three weeksinterval [AB(E)CD], [AB(E)CD]₂ or [AB(E)CD]₃ oligosaccharides conjugatedto tetanus toxoid (10 μg oligosaccharide/mice/injection). Control micereceived detoxified LPS from S. flexneri 2a conjugated to tetanus toxoid(10 μg polysaccharide/mice/injection) by multipoint attachment, asdescribed by Robbins J. B. (J. Infect. Dis. 161: 821-832), or tetanustoxoid alone (140 μg/mice/injection), following the same immunizationschedule. Seven days after the last injection, the mice received a lastboost of conjugates, in the same conditions.

2) Antibody Response Analysis

The anti-LPS 2a, anti-oligosaccharides and anti-tetanus toxoid (TTantibody response was analysed by ELISA, seven days after the thirdimmunization (before the boost), and seven days after the boost.Microtiter plates were coated with the corresponding antigen incarbonate buffer pH 9.6, at a concentration of 5 μg/ml, for the LPS.Biotinylated oligosaccharide solutions were adjusted to equimolarconcentrations based on the amount of ligand present in the respectiveglycoconjugate and incubated with PBS/BSA 1% for 30 min at 4° C. Boundantibodies were detected by using peroxidase-conjugated anti-mouseimmunoglobulins. After washing with PBS-Tween 20 (0.05%), alkalinephosphatase-conjugated anti-mouse IgG was added at a dilution of 1:5000(SIGMA) for 1 h at 37° C. After washing with PBS-Tween 20 (0.05%), thesubstrate was added (12 mg of p-nitrophenylphosphate in 1.2 ml of Tris,HCl buffer ph 8.8 and 10.8 ml of NaCl 5M). Once the color developed, theplate was read at 405 nm (Dinatech MR 4000 microplate reader). Antibodytiters were defined as the last dilution of the sample giving an OD atleast twice that of the control.

3) Protection Studies

The mice immunized i.m. were challenged i.n. with 10⁸ virulent bacteria8 days after the boost. Measurement of bacterial load was performed 24 hpost-infection, as described in example II.

B) Results

The anti-LPS 2a, anti-oligosaccharides and anti-tetanus toxoid (TT)antibody response was analysed by ELISA, seven days after the thirdimmunization (before the boost), and seven days after the boost.

TABLE H: Anti-LPS 2a antibody response induced by tetra-andhexasaccharides conjugates J7 after third immunization (1) and J7 afterboost (2) TETRA HEXA TT LPS/TT Mouse anti-LPS anti-LPS anti-LPS anti-LPSn° (1) (2) (1) (2) (1) (2) (1) (2) 1 0 0 0 0 0 0 400 1600 2 0 0 0 0 0 00 400 3 0 0 0 0 0 0 0 800 4 0 0 0 0 0 0 0 200 5 0 0 0 0 0 0 0 200 6 0 00 0 0 0 0 0 7 0 0 0 0 0 0 0 0

TABLE I: Anti-LPS 2a antibody response induced by penta-, deca- andpentadccasaccharides conjugates J7 after third immunization (1) and J7after boost (2) PENTA DECA PENTADECA Anti-LPS Anti-LPS Anti-LPS Mouse n°(1) (2) (1) (2) (1) (2) 1 0 6400 3200 12800 6400 12800 2 0 3200 320025600 12800 51200 3 0 400 800 800 12800 51200 4 0 200 1600 3200 2560025600 5 0 0 400 1600 25600 51200 6 0 0 25600 400 6400 12800 7 0 0 0 8003200 3200 8 0 0 0 3200 12800 25600 9 0 0 0 6400 6400 25600 10 0 0 012800 12800 25600 11 0 0 0 3200 6400 25600 12 0 0 0 800 12800 25600 13 00 0 0 6400 12800 14 0 0 0 0 3200 12800 Percentage 0% 28.50% 42.85%85.70% 100% 100% or responders Mean of 1/728 1/2500 1/5200 1/110001/26000 antibody titers

TABLE J Anti-oligosaccharide antibody response induced by tetra-andhexasaccharides conjugates J7 after third immunization (1) and J7 afterboost (2) TETRA HEXA Mouse n° (1) (2) (1) (2) 1 51200 51200 200 800 251200 51200 200 6400 3 51200 51200 0 12800 4 12800 51200 0 0 5 0 51200 00 6 0 200 0 0 7 0 0 0 0 Mean of 1/24000 1/36600 1/50 1/3000 antibodytiters

TABLE K: anti-oligosaccharide antibody response induced by penta-, deca-and pentadecasaccharides conjugates J7 after third immunization (1) andJ7 after boost (2) PENTA DECA PENTADECA Mouse n° (1) (2) (1) (2) (1) (2)1 800 6400 200 800 3200 12800 2 1600 12800 100 25600 6400 51200 3 2001600 100 6400 6400 51200 4 800 12800 3200 800 6400 51200 5 800 1600 0400 6400 102400 6 0 12800 0 12800 1600 12800 7 0 25600 0 3200 6400 512008 0 6400 0 800 3200 51200 9 0 6400 0 600 3200 51200 10 0 25600 0 640012800 51200 11 0 800 0 200 3200 51200 12 0 1600 0 0 400 51200 13 0 64000 0 3200 51200 14 0 25600 0 0 800 51200 Mean of 1/300 1/10500 1/2501/4500 1/4500 1/49000 antibody titers

No anti-LPS antibodies are observed in the mice immunized with thetetra- and hexasacacharides conjugates despite of ananti-oligosaccharide antibody response.

Low levels of anti-LPS antibodies are observed in the mice immunizedwith the detoxified LPS conjugate.

High levels of anti-LPS antibodies are observed in the mice immunizedwith the penta-, deca and penta decasaccharides conjugates. However, theantibody response is improved with the longer oligosaccharide(pentadecasaccharide); after the third immunization 100% of the micereceiving the pentadecapeptides present anti-LPS antibodies, as comparedwith 85% and 30% only, for the mice receiving the decasaccharide and thepentasaccharide, respectively. Moreover, the anti-LPS antibody titers aswell as the homogeneity of the antibody response is higher in the miceimmunized with the pentadecasaccharide.

2) Protection Studies

The ability of the antibodies induced by immunization with theoligosaccharides-TT conjugate to protect against Shigella infection wasassayed by active protection studies in the mouse model of pulmonaryinfection.

Protection as assessed by a reduction of the bacteria load, was observedwith the penta, deca and pentadecasaccharides conjugates whereas neitherthe tetra- and hexa saccharides conjugates, nor the detoxified LPSconjugate induced protection (FIG. 34).

The invention claimed is:
 1. A conjugate molecule consisting of anoligo- or polysaccharide selected from the group consisting of:{AB(E)CD}n wherein: A is an alphaLRhap-(1,2) residue B is analphaLRhap-(1,3) residue C is an alphaLRhap-(1,3) residue E is analphaDGlcp-(1,4) residue D is a betaDGlcNAcp-(1,2) residue E is branchedto C and wherein n is an integer selected from 2, 3, covalently bound toa carrier.
 2. A molecule according to claim 1 wherein the carrier isselected among a protein or a peptide comprising at least one T-cellepitope, or a derivative thereof, which is recognized by T-cells and isable to induce an antibody response.
 3. A molecule according to claim 2,wherein the carrier is the peptide PADRE.
 4. A molecule according toclaim 2, wherein the carrier is the tetanus toxoid.
 5. A moleculeaccording to claim 1, wherein the carrier is biotin.
 6. A moleculeaccording to claim 1, wherein the saccharide is directly bound to thecarrier.
 7. A molecule according to claim 1, wherein the saccharide isbound to the carrier via a spacer.
 8. A molecule according to claim 1,wherein the saccharide to carrier ratio is comprised between 1:1 and30:1.
 9. An immunogenic composition comprising a molecule according toanyone of claims 1, 2, 4, or 6-8 and a physiologically acceptablevehicle.
 10. The composition of claim 9 further comprising an immunogenwhich affords protection against pathogens responsible for diarrhoealdisease in humans.
 11. The composition of claim 10, which is formulatedfor parenteral, oral or intranasal administration.
 12. A kit for thediagnostic of Shigella flexneri type 2a infection, wherein said kitcomprises a molecule according to claim 1.